# Impaired excitability of fast-spiking neurons in a novel mouse model of  KCNC1  epileptic encephalopathy

<!-- wengert-2026-kcnc1 · claim assignments as tika v2 notes · &#x27E6;&gt;author claim=KEY: @{span} what the claim says&#x27E7; · KEY is the claim's UUID, or `gap` (a result no claim accounts for), or `no-assertion` (the span states no result). An unmarked sentence carries no result and was never an obligation. -->


## abstract

The recurrent pathogenic variant KCNC1- p.Ala421Val (A421V) is a cause of developmental and epileptic encephalopathy characterized by moderate-to-severe developmental delay/intellectual disability, and infantile-onset treatment-resistant epilepsy with multiple seizure types, including myoclonic seizures.

Yet, the mechanistic basis of this disease, and of the KCNC1 disease spectrum, remains unclear.

KCNC1 encodes Kv3.1, a voltage-gated potassium channel subunit that is strongly and selectively expressed in neurons capable of generating action potentials at high frequency, including parvalbumin-positive fast-spiking GABAergic inhibitory interneurons in cerebral cortex (PV-INs) that are known to be important for cognitive function and plasticity as well as control of network excitation to prevent seizures.

In this study, we generate a novel transgenic mouse model with conditional expression of the A421V pathogenic missense variant ( Kcnc1 -A421V/+ mice) to explore the specific physiological mechanisms of KCNC1 developmental and epileptic encephalopathy.

Our results indicate that global heterozygous expression of the A421V variant leads to cognitive impairment, epilepsy, and premature lethality.

We observe decreased PV-IN cell surface expression of Kv3.1 via immunohistochemistry, decreased voltage-gated potassium current density in PV-INs using outside-out nucleated macropatch recordings in brain slice, and profound impairments in the intrinsic excitability of cerebral cortex PV-INs (but not excitatory neurons) via current-clamp electrophysiology.

In vivo two-photon calcium imaging revealed altered activity in Kcnc1 -A421V/+ PV-INs and excitatory cells, as well as hypersynchronous discharges correlated with brief paroxysmal movements that were subsequently shown to be myoclonic seizures on electroencephalography.

We found alterations in PV-IN-mediated inhibitory neurotransmission in young adult but not juvenile Kcnc1 -A421V/+ mice relative to wild-type controls.

Together, these results establish the specific impact of the recurrent Kv3.1-A421V variant on neuronal excitability and synaptic physiology across development to drive network dysfunction underlying KCNC1 epileptic encephalopathy.


## introduction

Introduction Variants in KCNC1 , which encodes the voltage-gated potassium (K + ) channel subunit Kv3.1, cause KCNC1 -related neurological disorders, a spectrum of clinical phenotypes ranging from nonspecific intellectual disability to progressive myoclonus epilepsy and developmental and epileptic encephalopathy (DEE) ( Oliver et al., 2017 ; Cameron et al., 2019 ; Park et al., 2019 ; Li et al., 2021 ; Clatot et al., 2023 ; Feng et al., 2024 ).

Kv3.1 is one of four members (Kv3.1-Kv3.4) of the Kv3 subfamily of voltage-gated K + channels.

Kv3 channels show unique biophysical properties relative to other voltage-gated K + channels, including a depolarized voltage dependence of activation, fast rates of activation and deactivation, and little/no inactivation, properties that are exquisitely tuned to generate brief spikes and limit inter-spike interval, and thereby support rapid cycling required for reliable fast-spiking in Kv3-expressing neurons ( Weiser et al., 1995 ; Massengill et al., 1997 ; Sekirnjak et al., 1997 ; Gan and Kaczmarek, 1998 ; Martina et al., 1998 ; Wang et al., 1998 ; Erisir et al., 1999 ; Rudy and McBain, 2001 ; Lien and Jonas, 2003 ; Akemann and Knöpfel, 2006 ; Sacco et al., 2006 ; Martina et al., 2007 ).

Thus, Kv3 channels are highly and specifically expressed in cellular populations throughout the brain known to generate action potentials (APs) at high frequency, including cerebellar granule and Purkinje cells, neurons of the reticular thalamus, as well as parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) in the neocortex, hippocampus, amygdala, and basal ganglia ( Rudy et al., 1999 ; Kaczmarek and Zhang, 2017 ).

Alterations in Kv3.1 function would be expected to have a profound impact on neuronal excitability of fast-spiking neurons with downstream effects on circuits containing Kv3.1-expressing cells.

Our previous study using a novel mouse model of Progressive Myoclonus Epilepsy Type 7 (PME7 or EPM7) harboring the recurrent missense variant KCNC1- p.Arg320His (R320H) indicated that loss of Kv3.1 function alters excitability and synaptic neurotransmission in cerebral cortex PV-INs and cerebellar granule cells in adult heterozygous Kcnc1- p.R320H/+ mice ( Feng et al., 2024 ).

In contrast to EPM7, patients harboring de novo heterozygous KCNC1- p.Ala421Val (A421V) variants exhibit DEE, with moderate to severe developmental delay/intellectual disability without regression, variable but mild nonprogressive ataxia, and treatment-resistant epilepsy onset in infancy with multiple seizure types, including myoclonic seizures ( Oliver et al., 2017 ; Cameron et al., 2019 ; Park et al., 2019 ; Li et al., 2021 ).

Examination of the function of voltage-gated K + channels containing variant vs. wild-type (WT) Kv3.1 in heterologous systems has indicated that the A421V variant is a near-complete loss of function at the level of the channel, generating K + currents that are significantly reduced in magnitude relative to WT ( Cameron et al., 2019 ; Park et al., 2019 ).

Hence, while both the R320H and A421V variants are loss of function with a proposed dominant-negative action on tetrameric Kv3 channels composed of WT and variant subunits in heterologous systems, the A421V variant is a more severe loss of function, consistent with the associated clinical phenotype with earlier age of onset and treatment-resistant epilepsy.

The A421 residue is localized between the selectivity filter and the PVP motif of Kv3.1. Molecular modeling shows that the A421V variant does not lead to obvious steric hindrance in the channel, yet could possibly influence gating and selectivity through the addition of hydrophobic carbon atoms in the Kv3.1 pore ( Li et al., 2021 ).

Yet, the precise mechanisms underlying how the A421V variant impacts native neuronal Kv3 currents, neuronal physiology, and ultimately results in DEE, and how this differs from other disease-associated variants in KCNC1, remain unclear.

In this study, we generated a novel mouse model of KCNC1 DEE – Kcnc1 -Flox(p.Ala421Val)/+ (i.e. Kcnc1 -A421V/+) mice – to determine the impact of heterozygous expression of the Kcnc1- p.A421V variant as seen in patients on native voltage-gated K + channel currents, intrinsic excitability of Kv3.1-expressing neurons, inhibitory synaptic neurotransmission and function in cortical microcircuits, and epilepsy phenotype.

Our results indicate that global heterozygous expression of the Kcnc1 -p.A421V allele results in developmental impairment, cognitive dysfunction, epilepsy including prominent myoclonic seizures, and premature lethality due to seizure-induced sudden death.

Patch-clamp electrophysiological recordings demonstrate that Kv3-like voltage-gated K + current density is significantly reduced in PV-INs driven at least in part by impaired trafficking and cell surface expression of Kv3.1, with resulting alterations in AP waveform and impaired intrinsic excitability.

Excitatory cell physiology was unchanged in the Kcnc1 -A421V/+ mice, suggesting that the phenotype is related to inhibitory neuron dysfunction.

Investigation of synaptic neurotransmission revealed no significant differences between WT and Kcnc1 -A421V/+ PV-IN-mediated inhibitory neurotransmission at the early juvenile time window (postnatal day [P]16–21), but significantly altered properties at the young adult time point (P32–42), consistent with the observed progressive worsening of epilepsy in the mouse model and suggesting that altered Kv3.1 function leads to impairments in PV-IN synaptic function in a developmentally regulated manner.

Overall, these results indicate that the Kcnc1 -A421V variant is physiologically loss of function in native neurons with resulting impairment of intrinsic excitability and synaptic transmission of Kv3.1-expressing parvalbumin-positive fast-spiking cells, yielding epilepsy and cognitive impairment.


## results

Results Generation of the Kcnc1-A421V/+ mouse model of KCNC1 epilepsy We generated a novel transgenic mouse (see Materials and methods) that conditionally expresses Kcnc1- p.A421V/+ ( Kcnc1- p.A421V/+ mice) homologous to a recurrent KCNC1 variant previously identified in human patients with DEE ( Oliver et al., 2017 ; Cameron et al., 2019 ; Park et al., 2019 ).

Briefly, the Kcnc1 c.1262C>T missense variant was introduced into an ES cell line via gene targeting, converting a GCT to GTT and leading to the Ala421Val amino acid change.

A targeting vector containing part of intron 1 followed by the coding sequence of exons 2–4 flanked by loxP sites was then introduced upstream of the modified endogenous sequence ( Figure 1A ).⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{A targeting vector containing part of intron 1 followed by the coding sequence of exons 2–4 flanked by loxP sites was then introduced upstream of the modified endogenous sequence ( Figure 1A ).} scope-a421v-knockin-mouse — The scope claim defines the experimental envelope as a novel transgenic mouse conditionally expressing Kcnc1-A421V, which is exactly the targeting design described here.⟧

Thus, in the absence of Cre recombinase, there is expression of the introduced 5’ WT exons 2–4; in the presence of Cre recombinase, there is Cre-mediated excision of the floxed WT exons 2–4 coding sequence and expression of Kcnc1 harboring the c.1262C>T substitution, resulting in the single amino acid change p.Ala421Val ( Figure 1A ).⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{Thus, in the absence of Cre recombinase, there is expression of the introduced 5’ WT exons 2–4; in the presence of Cre recombinase, there is Cre-mediated excision of the floxed WT exons 2–4 coding sequence and expression of Kcnc1 harboring the c.1262C>T substitution, resulting in the single amino acid change p.Ala421Val ( Figure 1A ).} scope-a421v-knockin-mouse — The Cre-dependent switch from WT to A421V exons is the conditional-expression design the scope claim names.⟧

Sanger sequencing confirmed the knock-in missense mutation in exon 2, and subsequent PCR showed Cre-dependent genome recombination of the variant ( Figure 1B and C ).⟦>zach claim=gap: @{Sanger sequencing confirmed the knock-in missense mutation in exon 2, and subsequent PCR showed Cre-dependent genome recombination of the variant ( Figure 1B and C ).} No claim records the molecular validation of the knock-in — that Sanger sequencing confirmed the c.1262C>T substitution and PCR confirmed Cre-dependent recombination.⟧

We utilized a breeding strategy that allowed us to examine the behavior and physiology of mice expressing the Kcnc1 variant globally (via cross to Actb-Cre mice; JAX#: 003376), as is presumed the case with human patients harboring KCNC1- p. A421V as a de novo pathogenic variant ( Figure 1D ).⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{We utilized a breeding strategy that allowed us to examine the behavior and physiology of mice expressing the Kcnc1 variant globally (via cross to Actb-Cre mice; JAX#: 003376), as is presumed the case with human patients harboring KCNC1- p. A421V as a de novo pathogenic variant ( Figure 1D ).} scope-a421v-knockin-mouse — The scope claim explicitly states the global heterozygous configuration achieved by crossing to Actb-Cre.⟧

We also used a transgenic mouse line (C57BL/6-Tg(Pvalb-tdTomato)15Gfng/J; JAX#: 027395) which fluorescently labels PV-INs with the red fluorescent protein tdTomato driven by the endogenous parvalbumin promoter ( Figure 1D ).⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{We also used a transgenic mouse line (C57BL/6-Tg(Pvalb-tdTomato)15Gfng/J; JAX#: 027395) which fluorescently labels PV-INs with the red fluorescent protein tdTomato driven by the endogenous parvalbumin promoter ( Figure 1D ).} scope-a421v-knockin-mouse — The scope claim explicitly states the further cross to Pvalb-tdTomato for PV-IN identification.⟧

Our triple transgenic breeding strategy, therefore, produced experimental Kcnc1 -A421V/+ mice and WT littermates of both sexes containing Cre and with ~50% harboring the tdTomato allele to guide physiological experiments targeting PV-INs.

The overall survival curve demonstrated that Kcnc1 -A421V/+ mice (N=33) exhibited premature death relative to their WT littermates (N=46) with no Kcnc1 -A421V/+ mice surviving beyond 122 days (***p<0.001 by Mantel-Cox test; Figure 1E ).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{The overall survival curve demonstrated that Kcnc1 -A421V/+ mice (N=33) exhibited premature death relative to their WT littermates (N=46) with no Kcnc1 -A421V/+ mice surviving beyond 122 days (***p<0.001 by Mantel-Cox test; Figure 1E ).} a421v-mice-die-before-122d⟧

Figure 1. Design of a novel mouse model of KCNC1 developmental and epileptic encephalopathy.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Figure 1. Design of a novel mouse model of KCNC1 developmental and epileptic encephalopathy.} a421v-mice-die-before-122d⟧

( A ) Design and structure of the conditional Kcnc1 -A421V allele.⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{( A ) Design and structure of the conditional Kcnc1 -A421V allele.} scope-a421v-knockin-mouse — This panel depicts the conditional Kcnc1-A421V allele whose design the scope claim states.⟧

Upon Cre-mediated recombination, the inserted wild-type (WT) coding sequence (CDS) flanked by LoxP sites is removed and the A421V variant inserted into exon 2 is expressed.

( B ) Sequencing results indicate successful targeting of c.1262C>T to introduce the heterozygous A421V variant.⟦>zach claim=gap: @{( B ) Sequencing results indicate successful targeting of c.1262C>T to introduce the heterozygous A421V variant.} No claim records the sequencing validation that the c.1262C>T knock-in was successfully targeted.⟧

( C ) PCR confirmation of two HET founders ( Kcnc1 -A421V/+) and two WT littermates. 167 bp, WT allele fragment; 207 bp, floxed allele fragment.⟦>zach claim=gap: @{( C ) PCR confirmation of two HET founders ( Kcnc1 -A421V/+) and two WT littermates. 167 bp, WT allele fragment; 207 bp, floxed allele fragment.} No claim records the PCR genotyping that confirmed the heterozygous founders carry the floxed allele.⟧

( D ) Breeding strategy to generate control and experimental mice in which the Kcnc1 -A421V variant is expressed globally and PV cells are fluorescently labeled for targeted recording.⟦>zach claim=33883d00-c963-41b8-a13d-ca9e2cb8b1b2: @{( D ) Breeding strategy to generate control and experimental mice in which the Kcnc1 -A421V variant is expressed globally and PV cells are fluorescently labeled for targeted recording.} scope-a421v-knockin-mouse — The breeding strategy this panel depicts — global variant expression plus fluorescent PV labelling — is spelled out in the scope claim.⟧

( E ) Survival plot of WT (N=46; black ) and Kcnc1 -A421V/+ (N=33; green ) mice. ***p<0.001 by log-rank Mantel-Cox curve comparison.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{( E ) Survival plot of WT (N=46; black ) and Kcnc1 -A421V/+ (N=33; green ) mice. ***p<0.001 by log-rank Mantel-Cox curve comparison.} a421v-mice-die-before-122d⟧

Figure 1—figure supplement 1. Pvalb-tdTomato reporter effectively labels parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) in wild-type (WT) and Kcnc1 -A421V/+ mice.⟦>zach claim=gap: @{Figure 1—figure supplement 1. Pvalb-tdTomato reporter effectively labels parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) in wild-type (WT) and Kcnc1 -A421V/+ mice.} No claim records the validation of the Pvalb-tdTomato reporter as a faithful label for PV-INs in either genotype.⟧

( A ) Representative immunohistochemistry images for parvalbumin in WT (top row) and Kcnc1 -A421V/+ (bottom row) mice (postnatal day [P]21–33) showing a high degree of overlap between the tdTomato reporter and parvalbumin expression.⟦>zach claim=gap: @{( A ) Representative immunohistochemistry images for parvalbumin in WT (top row) and Kcnc1 -A421V/+ (bottom row) mice (postnatal day [P]21–33) showing a high degree of overlap between the tdTomato reporter and parvalbumin expression.} No claim records the immunohistochemical overlap between tdTomato and parvalbumin that validates the reporter.⟧

The asterisk indicates rare cells that are tdTomato + , but parvalbumin – .

The arrowhead indicates cells that are parvalbumin + but tdTomato – .

Scale bar, 100 μm.

( B ) Counts of PV cells per unit area (mm 2 ) are not different between WT and Kcnc1 -A421V/+ mice (N=3 mice/genotype).⟦>zach claim=gap: @{( B ) Counts of PV cells per unit area (mm 2 ) are not different between WT and Kcnc1 -A421V/+ mice (N=3 mice/genotype).} No claim records that neocortical PV cell density is unchanged in Kcnc1-A421V/+ mice.⟧

( C ) Sensitivity rate (probability of a cell being tdTomato + if it is parvalbumin + ; N = 6 mice/genotype).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( C ) Sensitivity rate (probability of a cell being tdTomato + if it is parvalbumin + ; N = 6 mice/genotype).} a421v-kv31-membrane-trafficking-impaired⟧

( D ) False-positive probability (proportion of all tdTomato + cells that are parvalbumin negative; N = 6 mice/genotype).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) False-positive probability (proportion of all tdTomato + cells that are parvalbumin negative; N = 6 mice/genotype).} a421v-kv31-membrane-trafficking-impaired⟧

Figure 1—figure supplement 2. Early postnatal development of Kcnc1 -A421V/+ mice.⟦>zach claim=b7a7c44d-b512-45b8-a790-a1b2c3b216e7: @{Figure 1—figure supplement 2. Early postnatal development of Kcnc1 -A421V/+ mice.} a421v-weight-reduced-milestones-normal⟧

( A ) Representative example image showing littermate wild-type (WT) and Kcnc1 -A421V/+ mice at postnatal day 21. ( B ) Average body weights for WT (N=10; black ) and Kcnc1 -A421V/+ (N=11; green ) at postnatal days 7, 14, and 21. ( C ) Average brain weights for WT and Kcnc1 -A421V/+ mice at postnatal days 7 (WT, N=2; Kcnc1 -A421V/+, N=6), 14 (WT, N=3; Kcnc1 -A421V/+, N=3), and 21 (WT, N=4; Kcnc1 -A421V/+, N=6).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( A ) Representative example image showing littermate wild-type (WT) and Kcnc1 -A421V/+ mice at postnatal day 21. ( B ) Average body weights for WT (N=10; black ) and Kcnc1 -A421V/+ (N=11; green ) at postnatal days 7, 14, and 21. ( C ) Average brain weights for WT and Kcnc1 -A421V/+ mice at postnatal days 7 (WT, N=2; Kcnc1 -A421V/+, N=6), 14 (WT, N=3; Kcnc1 -A421V/+, N=3), and 21 (WT, N=4; Kcnc1 -A421V/+, N=6).} a421v-kv31-membrane-trafficking-impaired⟧

Note that for both B and C, error bars depicting SEM are present, but do not appear beyond the symbols.

( D ).⟦>zach claim=no-assertion: @{( D ).} A bare panel label with no content of its own.⟧

Onset of developmental and motor benchmarks for WT (N=7) and Kcnc1 -A421V/+ (N=10) mice.⟦>zach claim=b7a7c44d-b512-45b8-a790-a1b2c3b216e7: @{Onset of developmental and motor benchmarks for WT (N=7) and Kcnc1 -A421V/+ (N=10) mice.} a421v-weight-reduced-milestones-normal — This panel displays the developmental and motor benchmark onsets the claim reports as unaltered.⟧

( E ) Onset of startle reflex and other typical behavioral/motor milestones in WT (N=7) and Kcnc1 -A421V/+ (N=10) mice. ***p<0.001 by mixed-effects analysis followed by Sidak’s multiple comparisons post hoc test.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{( E ) Onset of startle reflex and other typical behavioral/motor milestones in WT (N=7) and Kcnc1 -A421V/+ (N=10) mice. ***p<0.001 by mixed-effects analysis followed by Sidak’s multiple comparisons post hoc test.} a421v-mice-die-before-122d⟧

Counts of PV-INs per unit of neocortical area were not different between WT and Kcnc1 -A421V/+ mice (P24–33), indicating that expression of A421V did not alter the density of neocortical PV-INs ( Figure 1—figure supplement 1A and B ), consistent with the fact that interneuron migration is complete prior to appreciable expression of Kcnc1 in mouse.⟦>zach claim=gap: @{Counts of PV-INs per unit of neocortical area were not different between WT and Kcnc1 -A421V/+ mice (P24–33), indicating that expression of A421V did not alter the density of neocortical PV-INs ( Figure 1—figure supplement 1A and B ), consistent with the fact that interneuron migration is complete prior to appreciable expression of Kcnc1 in mouse.} No claim records that PV-IN density per unit neocortical area is unaltered by A421V expression.⟧

We separately used immunohistochemistry to validate our genetic strategy for labeling PV-INs ( Figure 1—figure supplement 1 ): The average sensitivity rates were greater than 0.8 in both groups as previously reported ( Kaiser et al., 2016 ) and were not different by genotype ( Figure 1—figure supplement 1 ), with the average false-positive identification rate less than 0.1 for each group ( Figure 1—figure supplement 1 ).⟦>zach claim=gap: @{We separately used immunohistochemistry to validate our genetic strategy for labeling PV-INs ( Figure 1—figure supplement 1 ): The average sensitivity rates were greater than 0.8 in both groups as previously reported ( Kaiser et al., 2016 ) and were not different by genotype ( Figure 1—figure supplement 1 ), with the average false-positive identification rate less than 0.1 for each group ( Figure 1—figure supplement 1 ).} No claim records the sensitivity and false-positive rates that validate the tdTomato labelling strategy.⟧

Behavioral testing of Kcnc1 -A421V/+ mice Kcnc1 -A421V/+ mice underwent an assessment of developmental milestones at P5–15, as done previously ( Feng et al., 2024 ).

Although Kcnc1 -A421V/+ mice exhibited reduced body ( Figure 1—figure supplement 2A and B ) and brain ( Figure 1—figure supplement 2C ) weights relative to their WT littermates – as seen previously with Kcnc1 knockout mice ( Ho et al., 1997 ) – we did not detect other developmental abnormalities in the onset of fur appearance, eye opening, ear canal opening, incisor eruption, head elevation, shoulder elevation, auditory startle, horizontal screen test, vertical screen test, cliff avoidance, quadruple walking, and negative geotaxis ( Figure 1—figure supplement 2D and E ).⟦>zach claim=b7a7c44d-b512-45b8-a790-a1b2c3b216e7: @{Although Kcnc1 -A421V/+ mice exhibited reduced body ( Figure 1—figure supplement 2A and B ) and brain ( Figure 1—figure supplement 2C ) weights relative to their WT littermates – as seen previously with Kcnc1 knockout mice ( Ho et al., 1997 ) – we did not detect other developmental abnormalities in the onset of fur appearance, eye opening, ear canal opening, incisor eruption, head elevation, shoulder elevation, auditory startle, horizontal screen test, vertical screen test, cliff avoidance, quadruple walking, and negative geotaxis ( Figure 1—figure supplement 2D and E ).} a421v-weight-reduced-milestones-normal⟧

These results suggest that while body and brain weights are reduced, the Kcnc1 -A421V/+ mice show otherwise typical gross anatomical and functional development within the early developmental time point examined (P5–15), as determined by and within the sensitivity of the tests readily available for such evaluation.

We next tested whether cognitive function was altered in young adult Kcnc1 -A421V/+ mice (P35–65).

To this end, we assessed spatial memory in juvenile Kcnc1 -A421V/+ mice in the Barnes maze task ( Figure 2A ).⟦>zach claim=no-assertion: @{To this end, we assessed spatial memory in juvenile Kcnc1 -A421V/+ mice in the Barnes maze task ( Figure 2A ).} Narration of which task was run, reporting no result.⟧

Kcnc1 -A421V/+ mice showed a significant delay compared to controls in the acquisition of the escape hole position ( Figure 2A and B ).⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{Kcnc1 -A421V/+ mice showed a significant delay compared to controls in the acquisition of the escape hole position ( Figure 2A and B ).} a421v-spatial-learning-working-memory-impaired⟧

This defect was most pronounced by significantly longer escape latencies during the second day of acquisition trials, suggesting an impairment in spatial learning.

During the probe trial, conducted in the absence of the escape box (to assess memory retention), Kcnc1 -A421V/+ mice spent the same time as WT littermates in the target quadrant ( Figure 2C ), indicating intact long-term memory of the escape hole position.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{During the probe trial, conducted in the absence of the escape box (to assess memory retention), Kcnc1 -A421V/+ mice spent the same time as WT littermates in the target quadrant ( Figure 2C ), indicating intact long-term memory of the escape hole position.} a421v-spatial-learning-working-memory-impaired⟧

Figure 2. Impaired cognitive function in Kcnc1 -A421V/+ mice.⟦>zach claim=a2af2706-be34-44a7-b65b-6a11ae98c2a8: @{Figure 2. Impaired cognitive function in Kcnc1 -A421V/+ mice.} Inhibitory dysfunction in Kcnc1-A421V/+ mice emerges progressively by young adulthood.⟧

( A ) Example pathways for wild-type (WT) (black) and Kcnc1 -A421V/+ (green) during the 4-day acquisition phase of the Barnes maze test.⟦>zach claim=no-assertion: @{( A ) Example pathways for wild-type (WT) (black) and Kcnc1 -A421V/+ (green) during the 4-day acquisition phase of the Barnes maze test.} A representative-example panel label explaining the colour coding, not a finding.⟧

Blue dot indicates the escape hole.

( B ) Average escape latency during the acquisition phase for WT (black; N=15) and Kcnc1 -A421V/+ (green; N=13) mice.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{( B ) Average escape latency during the acquisition phase for WT (black; N=15) and Kcnc1 -A421V/+ (green; N=13) mice.} a421v-spatial-learning-working-memory-impaired⟧

( C ) Group data for time spent in target quadrant during probe trial of Barnes maze.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{( C ) Group data for time spent in target quadrant during probe trial of Barnes maze.} a421v-spatial-learning-working-memory-impaired⟧

(D–F) Group data for WT (black; N=16) and Kcnc1 -A421V/+ mice (green; N=15) during the Y-maze test for spatial working memory.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{(D–F) Group data for WT (black; N=16) and Kcnc1 -A421V/+ mice (green; N=15) during the Y-maze test for spatial working memory.} a421v-spatial-learning-working-memory-impaired⟧

(D) Spontaneous alternation percentage.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{(D) Spontaneous alternation percentage.} a421v-spatial-learning-working-memory-impaired⟧

( E ) Total number of arm entries.⟦>zach claim=no-assertion: @{( E ) Total number of arm entries.} A bare panel label naming the quantity plotted.⟧

( G ) Total distance traveled.⟦>zach claim=no-assertion: @{( G ) Total distance traveled.} A bare panel label naming the quantity plotted.⟧

Data are shown as mean ± SEM and were analyzed by two-way repeated-measures ANOVA with Tukey’s post hoc test ( B ) and unpaired t-test ( D–F ).⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{Data are shown as mean ± SEM and were analyzed by two-way repeated-measures ANOVA with Tukey’s post hoc test ( B ) and unpaired t-test ( D–F ).} a421v-spatial-learning-working-memory-impaired⟧

Significance is denoted as *p<0.05 or **p<0.01. We then assessed spatial working memory using the Y maze spontaneous alternation test ( Figure 2D–F ).⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Significance is denoted as *p<0.05 or **p<0.01. We then assessed spatial working memory using the Y maze spontaneous alternation test ( Figure 2D–F ).} pv-in-inhibitory-synapse-altered-adult⟧

Spontaneous alternation is a behavior driven by the innate tendency of rodents to alternate between recently visited arms to explore previously unvisited areas of the maze.

Relative to WT littermates, Kcnc1 -A421V/+ displayed a statistically significant decrease in the percentage of spontaneous alternations ( Figure 2D ).⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{Relative to WT littermates, Kcnc1 -A421V/+ displayed a statistically significant decrease in the percentage of spontaneous alternations ( Figure 2D ).} a421v-spatial-learning-working-memory-impaired⟧

Importantly, the total number of arm entries ( Figure 2E ) and the distance traveled ( Figure 2F ) did not differ between genotypes, suggesting that the observed deficit was not due to differences in general activity, motor function, or exploratory drive.⟦>zach claim=3ea91341-e08d-4f0b-86fc-604dd4b1ebd6: @{Importantly, the total number of arm entries ( Figure 2E ) and the distance traveled ( Figure 2F ) did not differ between genotypes, suggesting that the observed deficit was not due to differences in general activity, motor function, or exploratory drive.} prediction-cognitive-deficits — The prediction requires the cognitive deficit to be specific and not attributable to locomotor or exploratory confounds, which is exactly what these unchanged activity measures establish.⟧

Taken together, these behavioral observations indicate deficits in both spatial learning and working memory systems in young adult Kcnc1 -A421V/+ mice.

Kcnc1 -A421V/+ mice exhibit reduced voltage-gated potassium channel currents and altered Kv3.1 expression Previous studies in heterologous expression systems have reported that the A421V variant is physiologically loss of function and generates strongly attenuated voltage-gated K + channel currents in Xenopus laevis oocytes ( Cameron et al., 2019 ; Park et al., 2019 ), albeit with conflicting conclusions related to the presence of a dominant-negative action.

In our recordings of HEK cells expressing WT and A421V Kv3.1 subunits, A421V was a profound loss of function, although a small magnitude K + current was detectable (which cannot be easily distinguished from the small endogenous delayed rectified potassium current present in HEK cells; Figure 3—figure supplement 1 ).⟦>zach claim=gap: @{In our recordings of HEK cells expressing WT and A421V Kv3.1 subunits, A421V was a profound loss of function, although a small magnitude K + current was detectable (which cannot be easily distinguished from the small endogenous delayed rectified potassium current present in HEK cells; Figure 3—figure supplement 1 ).} No claim records the heterologous HEK-cell result that A421V alone is a profound loss of function.⟧

A 50:50 mixture of WT and A421V subunits, more approximating the clinical condition, produced K + currents that were ~42% of the magnitude and slightly shifted in the hyperpolarized direction relative to the WT condition ( Figure 3—figure supplement 1D and E ).⟦>zach claim=gap: @{A 50:50 mixture of WT and A421V subunits, more approximating the clinical condition, produced K + currents that were ~42% of the magnitude and slightly shifted in the hyperpolarized direction relative to the WT condition ( Figure 3—figure supplement 1D and E ).} No claim records the HEK-cell co-expression result — ~42% of WT current magnitude with a hyperpolarizing shift for the 50:50 WT/A421V mixture.⟧

Considering that Kv3.1 channels form heterotetramers with other Kv3 channel isoforms (likely Kv3.2) in cerebral cortex PV-INs, we also sought to clarify the impact of the A421V Kcnc1 variant on native neuronal voltage-gated K + channel function in neocortical layer II-IV PV-INs, known to express high levels of Kv3.1 ( Chow et al., 1999 ), from Kcnc1 -A421V/+ mice at the juvenile timepoint of P16–21. The outside-out nucleated macropatch technique allowed for high-quality electrophysiological recordings of somatic voltage-gated K + currents ( Figure 3A–C ).⟦>zach claim=no-assertion: @{Considering that Kv3.1 channels form heterotetramers with other Kv3 channel isoforms (likely Kv3.2) in cerebral cortex PV-INs, we also sought to clarify the impact of the A421V Kcnc1 variant on native neuronal voltage-gated K + channel function in neocortical layer II-IV PV-INs, known to express high levels of Kv3.1 ( Chow et al., 1999 ), from Kcnc1 -A421V/+ mice at the juvenile timepoint of P16–21. The outside-out nucleated macropatch technique allowed for high-quality electrophysiological recordings of somatic voltage-gated K + currents ( Figure 3A–C ).} Rationale and method narration introducing the macropatch recordings, with no result of its own.⟧

Relative to WT controls (n=13 cells, N=3 mice), the current density of voltage-gated K + currents in the Kcnc1 -A421V/+ mice (n=17 cells, N=3 mice) was markedly reduced across all voltages examined (***p<0.001; repeated-measures two-way ANOVA; Figure 3B–D ).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Relative to WT controls (n=13 cells, N=3 mice), the current density of voltage-gated K + currents in the Kcnc1 -A421V/+ mice (n=17 cells, N=3 mice) was markedly reduced across all voltages examined (***p<0.001; repeated-measures two-way ANOVA; Figure 3B–D ).} a421v-mice-die-before-122d⟧

Peak current densities were significantly lower in PV-INs from Kcnc1 -A421V/+ mice compared to WT controls (***p<0.001; t-test; Figure 3E ).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Peak current densities were significantly lower in PV-INs from Kcnc1 -A421V/+ mice compared to WT controls (***p<0.001; t-test; Figure 3E ).} a421v-mice-die-before-122d⟧

We did not observe differences in the voltage dependence ( Figure 3F ) or kinetics of activation ( Figure 3G ) when comparing voltage-gated K + channel currents from WT and Kcnc1 -A421V/+ mice.⟦>zach claim=f80417c3-ece7-4792-9e83-0b3f92d26ac2: @{We did not observe differences in the voltage dependence ( Figure 3F ) or kinetics of activation ( Figure 3G ) when comparing voltage-gated K + channel currents from WT and Kcnc1 -A421V/+ mice.} prediction-kv31-surface-expression-reduced — The prediction's corollary states that voltage-dependence of activation and activation kinetics of the residual current should be unchanged, which is the null result reported here.⟧

Together, these results demonstrate that PV-IN voltage-gated K + channel function is strongly impaired in the context of the Kcnc1 -p.A421V variant.

Figure 3. Parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from Kcnc1 -A421V/+ mice exhibit attenuated voltage-gated potassium channel currents and impaired membrane Kv3.1 expression.⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{Figure 3. Parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from Kcnc1 -A421V/+ mice exhibit attenuated voltage-gated potassium channel currents and impaired membrane Kv3.1 expression.} pv-ins-impaired-maximal-firing⟧

( A ) Representative image of a cell being recorded in the outside-out nucleated macropatch configuration.⟦>zach claim=no-assertion: @{( A ) Representative image of a cell being recorded in the outside-out nucleated macropatch configuration.} A representative image of the recording configuration, asserting nothing about genotype.⟧

( B–C ) Example family of traces of voltage-gated K + channel currents from a PV-IN from wild-type (WT) (B, black ) and Kcnc1 -A421V/+ (C, green ) mice (postnatal day [P]16–21).⟦>zach claim=no-assertion: @{( B–C ) Example family of traces of voltage-gated K + channel currents from a PV-IN from wild-type (WT) (B, black ) and Kcnc1 -A421V/+ (C, green ) mice (postnatal day [P]16–21).} Example traces with their colour coding, not a stated finding.⟧

( D ) Average voltage-gated K + channel current density for WT (n=13 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=17, N=3 mice).⟦>zach claim=b29d38ec-0fb8-4a7b-863e-470dd3eb378b: @{( D ) Average voltage-gated K + channel current density for WT (n=13 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=17, N=3 mice).} pv-ins-reduced-k-current-density — This panel displays the PV-IN voltage-gated K+ current density comparison that the claim reports as significantly reduced.⟧

( E ) Maximum K + channel current density per PV-IN macropatch in WT and Kcnc1 -A421V/+ mice.⟦>zach claim=b29d38ec-0fb8-4a7b-863e-470dd3eb378b: @{( E ) Maximum K + channel current density per PV-IN macropatch in WT and Kcnc1 -A421V/+ mice.} pv-ins-reduced-k-current-density — Peak K+ current density per macropatch is the same reduced-current finding the claim states.⟧

( F ) Averaged normalized plots of K + conductance relative to voltage command indicating voltage dependence of activation curves for WT and Kcnc1 -A421V/+ mice.⟦>zach claim=f80417c3-ece7-4792-9e83-0b3f92d26ac2: @{( F ) Averaged normalized plots of K + conductance relative to voltage command indicating voltage dependence of activation curves for WT and Kcnc1 -A421V/+ mice.} prediction-kv31-surface-expression-reduced — The activation curves this panel displays are the preserved voltage-dependence the prediction's corollary specifies.⟧

( G ) Average activation time constant for the voltage-gated K + channel currents relative to voltage command potential in both WT and Kcnc1 -A421V/+ mice.⟦>zach claim=f80417c3-ece7-4792-9e83-0b3f92d26ac2: @{( G ) Average activation time constant for the voltage-gated K + channel currents relative to voltage command potential in both WT and Kcnc1 -A421V/+ mice.} prediction-kv31-surface-expression-reduced — The activation time constants this panel displays are the preserved activation kinetics the prediction's corollary specifies.⟧

( H ) Representative images of individual cortical PV-INs from WT and Kcnc1 -A421V/+ mice stained for Kv3.1 (green).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( H ) Representative images of individual cortical PV-INs from WT and Kcnc1 -A421V/+ mice stained for Kv3.1 (green).} a421v-kv31-membrane-trafficking-impaired⟧

Plasma membrane (PM), nucleus (nuc), and cytosol (cyt) are indicated in the top left panel.

Note the markedly less pronounced Kv3.1 intensity in the plasma membrane in each of the Kcnc1 -A421V/+ examples and more prominent cytosolic labeling (presumably corresponding to endoplasmic reticulum).

( I ) Group quantification of ratio of membrane to cytosolic Kv3.1 for WT (n=49 cells, N=6 mice) and Kcnc1 -A421V/+ (n=48 cells, N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( I ) Group quantification of ratio of membrane to cytosolic Kv3.1 for WT (n=49 cells, N=6 mice) and Kcnc1 -A421V/+ (n=48 cells, N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

Mice were between the ages of P24 and P33. Data are shown as mean ± SEM or individual data points, and significance was determined using either repeated-measures two-way ANOVA or unpaired t-test where ***p<0.001. Figure 3—figure supplement 1. Loss of potassium current density in A421V-expressing HEK cells.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Mice were between the ages of P24 and P33. Data are shown as mean ± SEM or individual data points, and significance was determined using either repeated-measures two-way ANOVA or unpaired t-test where ***p<0.001. Figure 3—figure supplement 1. Loss of potassium current density in A421V-expressing HEK cells.} a421v-mice-die-before-122d⟧

( A–C ) Example Kv3.1 currents in HEK cells expressing wild-type (WT) (A; black), a 50:50 mixture of WT and A421V (B; blue), and A421V ( C ; green) Kv3.1 subunits.⟦>zach claim=no-assertion: @{( A–C ) Example Kv3.1 currents in HEK cells expressing wild-type (WT) (A; black), a 50:50 mixture of WT and A421V (B; blue), and A421V ( C ; green) Kv3.1 subunits.} Example traces with their colour coding, not a stated finding.⟧

Voltage command protocol shown below WT example.

( D ) Average current density for WT (black), WT+A421V (blue), and A421V (green).⟦>zach claim=gap: @{( D ) Average current density for WT (black), WT+A421V (blue), and A421V (green).} No claim records the HEK-cell current densities for WT, WT+A421V, and A421V that this panel carries.⟧

Note that the A421V variant leads to a profound loss of function.

( E ) Average normalized conductance relative to membrane potential for WT (black), WT+A421V (blue), and A421V (green).⟦>zach claim=gap: @{( E ) Average normalized conductance relative to membrane potential for WT (black), WT+A421V (blue), and A421V (green).} No claim records the HEK-cell conductance-voltage relations for the three subunit conditions.⟧

G-V curves for the A421V variant should be interpreted with caution as the recorded currents are exceedingly small and cannot be easily differentiated from the small endogenous delayed rectifier potassium currents known to be present in HEK cells.

The markedly decreased K + current magnitude observed in PV-INs without apparent alterations in gating properties is consistent with impaired conductance of the population of Kv3 channels, but could also be explained by impaired trafficking to the cell membrane.

To investigate this possibility, we examined Kv3.1 expression in PV-INs from juvenile (P24–33) WT and Kcnc1 -A421V/+ mice via immunohistochemistry.

Our results suggested that the amount of Kv3.1 at the membrane relative to cytosol was significantly altered in the Kcnc1 -A421V/+ mice (n=48 cells, N=5 mice) compared to WT controls (n=49 cells, N=6 mice; Figure 3H–I ).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{Our results suggested that the amount of Kv3.1 at the membrane relative to cytosol was significantly altered in the Kcnc1 -A421V/+ mice (n=48 cells, N=5 mice) compared to WT controls (n=49 cells, N=6 mice; Figure 3H–I ).} a421v-kv31-membrane-trafficking-impaired⟧

These findings support the conclusion that impaired trafficking to the cell surface at least contributes to the observed decrease in K + current in PV-INs in Kcnc1 -A421V/+ mice.

Intrinsic excitability of PV-INs is altered in Kcnc1 -A421V/+ mice We next examined intrinsic neuronal excitability in PV-INs in somatosensory neocortex layers II-IV to determine the impact of the voltage-gated K + channel dysfunction across two age ranges, juvenile (P16–21; Figure 4A–E ) and young adult mice (P32–42; Figure 4F–J ).⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Intrinsic excitability of PV-INs is altered in Kcnc1 -A421V/+ mice We next examined intrinsic neuronal excitability in PV-INs in somatosensory neocortex layers II-IV to determine the impact of the voltage-gated K + channel dysfunction across two age ranges, juvenile (P16–21; Figure 4A–E ) and young adult mice (P32–42; Figure 4F–J ).} pv-in-ap-waveform-altered-downstroke-apd50⟧

We performed whole-cell current-clamp recordings to generate a detailed comparison of passive membrane properties, properties of individual APs, and of repetitive firing, for PV-INs from primary somatosensory neocortex in WT vs. Kcnc1- A421V/+ mice at both time points ( Figure 4 and Tables 1 and 2 ).⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{We performed whole-cell current-clamp recordings to generate a detailed comparison of passive membrane properties, properties of individual APs, and of repetitive firing, for PV-INs from primary somatosensory neocortex in WT vs. Kcnc1- A421V/+ mice at both time points ( Figure 4 and Tables 1 and 2 ).} pv-in-ap-waveform-altered-downstroke-apd50⟧

PV-INs from both genotypes generated trains of repetitive APs in response to depolarizing current injection; however, frequency was profoundly reduced in Kcnc1 -A421V/+ mice relative to the WT control PV-INs at all current magnitudes examined (***p<0.001, repeated-measures two-way ANOVA; Figure 4B–D ) regardless of whether resting membrane potential was normalized across cells with DC bias current ( Figure 4—figure supplement 1 ).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{PV-INs from both genotypes generated trains of repetitive APs in response to depolarizing current injection; however, frequency was profoundly reduced in Kcnc1 -A421V/+ mice relative to the WT control PV-INs at all current magnitudes examined (***p<0.001, repeated-measures two-way ANOVA; Figure 4B–D ) regardless of whether resting membrane potential was normalized across cells with DC bias current ( Figure 4—figure supplement 1 ).} a421v-mice-die-before-122d⟧

When examining the properties of individual APs, Kcnc1 -A421V/+ and WT PV-INs showed marked differences in downstroke velocity and half-maximal AP duration (APD50), properties that are determined by Kv3 channel function.

AP amplitude was elevated in the Kcnc1 -A421V/+ mice at juvenile (P16–21; Figure 4E ; Table 1 ) and adult (P32–42; Figure 4J ; Table 2 ) time points, but only reached significance at P32–42 (***p<0.001, unpaired t-test).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{AP amplitude was elevated in the Kcnc1 -A421V/+ mice at juvenile (P16–21; Figure 4E ; Table 1 ) and adult (P32–42; Figure 4J ; Table 2 ) time points, but only reached significance at P32–42 (***p<0.001, unpaired t-test).} a421v-mice-die-before-122d⟧

Overall, PV-INs from Kcnc1- A421V/+ mice exhibited specific impairments consistent with reduction in Kv3 current, including altered properties of individual APs leading to a reduction in firing frequency, with a relative preservation of passive membrane properties not thought to be directly regulated by Kv3 channels.

Figure 4. Impaired parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) intrinsic excitability in juvenile and adult Kcnc1-A421V/+ mice.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Figure 4. Impaired parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) intrinsic excitability in juvenile and adult Kcnc1-A421V/+ mice.} pv-in-ap-waveform-altered-downstroke-apd50⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical PV-IN recorded in the whole-cell configuration to characterize intrinsic excitability.⟦>zach claim=no-assertion: @{( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical PV-IN recorded in the whole-cell configuration to characterize intrinsic excitability.} A representative image of the recorded cell, asserting nothing about genotype.⟧

( B–C ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA.⟦>zach claim=no-assertion: @{( B–C ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA.} Example traces with their colour coding and current steps, not a stated finding.⟧

The inset shows an expanded view of APs generated in response to the 400 pA current injection in both genotypes.

( D ) Average relationship between PV-IN AP frequency in response to a range of current injections for juvenile WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=36 cells, N = 12 mice).⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{( D ) Average relationship between PV-IN AP frequency in response to a range of current injections for juvenile WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=36 cells, N = 12 mice).} pv-ins-impaired-maximal-firing — This panel displays the juvenile PV-IN frequency-current relationship the claim reports as impaired.⟧

( E ) Representative overlaid examples of single APs and the corresponding phase plots for juvenile WT ( black ) and Kcnc1 -A421V/+ ( green ) PV-INs.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{( E ) Representative overlaid examples of single APs and the corresponding phase plots for juvenile WT ( black ) and Kcnc1 -A421V/+ ( green ) PV-INs.} pv-in-ap-waveform-altered-downstroke-apd50⟧

(F) Representative images for a layer IV neocortical PV-IN from an adult mouse.⟦>zach claim=no-assertion: @{(F) Representative images for a layer IV neocortical PV-IN from an adult mouse.} A representative image of the recorded cell, asserting nothing about genotype.⟧

( G–H ) Representative example traces displaying intrinsic excitability in adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.⟦>zach claim=no-assertion: @{( G–H ) Representative example traces displaying intrinsic excitability in adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.} Example traces from adult recordings, with no result stated.⟧

Inset shows an expanded view of APs induced by the 400 pA current step.

( I ) Average relationship between PV-IN AP frequency and current injection for adult WT (n = 14 cells, N = 3 mice) and Kcnc1 -A421V/+ mice (n=17 cells, N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( I ) Average relationship between PV-IN AP frequency and current injection for adult WT (n = 14 cells, N = 3 mice) and Kcnc1 -A421V/+ mice (n=17 cells, N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

( J ).

Representative overlaid examples of single APs and the corresponding phase plots for adult WT ( black ) and Kcnc1 -A421V/+ ( green ) PV-INs.

Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.} a421v-mice-die-before-122d⟧

Figure 4—figure supplement 1. Intrinsic physiology of neocortical layer II-IV parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) remains impaired when resting membrane potential is not normalized.⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{Figure 4—figure supplement 1. Intrinsic physiology of neocortical layer II-IV parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) remains impaired when resting membrane potential is not normalized.} pv-ins-impaired-maximal-firing — The claim states that PV-IN maximal firing is impaired, which is the effect this supplement reports as surviving without membrane-potential normalization.⟧

( A–B ) Representative example traces showing spiking in layer V neocortical PV-INs from wild-type (WT) (A, black) and Kcnc1 -A421V/+ ( B , green) mice in response to depolarizing current injections.⟦>zach claim=no-assertion: @{( A–B ) Representative example traces showing spiking in layer V neocortical PV-INs from wild-type (WT) (A, black) and Kcnc1 -A421V/+ ( B , green) mice in response to depolarizing current injections.} Example traces with their colour coding, not a stated finding.⟧

( C ) Average PV-IN spiking frequency relative to current injection.⟦>zach claim=d6e81203-d9f1-4344-97d0-48ec7b6afe39: @{( C ) Average PV-IN spiking frequency relative to current injection.} layer-v-pv-ins-subtle-impairment — This panel displays the layer V PV-IN spiking-frequency relationship whose genotype-by-current interaction the claim reports.⟧

A significant interaction effect between genotype and current injection was observed (***p<0.001) by two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{A significant interaction effect between genotype and current injection was observed (***p<0.001) by two-way ANOVA.} a421v-mice-die-before-122d⟧

Data are shown as mean ± SEM, and significance of post hoc comparisons (*p<0.05) by repeated-measures two-way ANOVA followed by Sidak’s multiple comparisons test.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM, and significance of post hoc comparisons (*p<0.05) by repeated-measures two-way ANOVA followed by Sidak’s multiple comparisons test.} pv-in-inhibitory-synapse-altered-adult⟧

Figure 4—figure supplement 2. Subtle abnormalities in neocortical layer V parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+.⟦>zach claim=d6e81203-d9f1-4344-97d0-48ec7b6afe39: @{Figure 4—figure supplement 2. Subtle abnormalities in neocortical layer V parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+.} layer-v-pv-ins-subtle-impairment⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a PV-positive cell in the reticular thalamus recorded in the whole-cell configuration to characterize intrinsic excitability.⟦>zach claim=no-assertion: @{( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a PV-positive cell in the reticular thalamus recorded in the whole-cell configuration to characterize intrinsic excitability.} A representative image of the recorded reticular thalamic cell, asserting nothing about genotype.⟧

( B ) Representative example traces of action potentials (APs) generated in response to current injections of varying magnitudes.⟦>zach claim=no-assertion: @{( B ) Representative example traces of action potentials (APs) generated in response to current injections of varying magnitudes.} Example traces of evoked action potentials, with no result stated.⟧

( C ) Average hyperpolarization-induced rebound APs in postnatal day (P)16–21 wild-type (WT) (n=16 cells, N=4 mice) and Kcnc1 -A421V/+ (n=19 cells, N=5 mice) in response to various current injections from 0 to –100 pA.⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( C ) Average hyperpolarization-induced rebound APs in postnatal day (P)16–21 wild-type (WT) (n=16 cells, N=4 mice) and Kcnc1 -A421V/+ (n=19 cells, N=5 mice) in response to various current injections from 0 to –100 pA.} a421v-kv31-membrane-trafficking-impaired⟧

( D ) Frequency-current relationship shows impaired intrinsic excitability in the RT PV cells relative to depolarizing current injections (0–400 pA).⟦>zach claim=37d23efe-7e34-48aa-82ff-b7f3ef45751c: @{( D ) Frequency-current relationship shows impaired intrinsic excitability in the RT PV cells relative to depolarizing current injections (0–400 pA).} rtn-neurons-impaired-excitability — The claim reports the attenuated frequency-current relationship in reticular thalamic PV neurons that this panel displays.⟧

Data are shown as mean ± SEM, and significance is denoted as *p<0.05 or ***p<0.001 by repeated-measures two-way ANOVA.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM, and significance is denoted as *p<0.05 or ***p<0.001 by repeated-measures two-way ANOVA.} pv-in-inhibitory-synapse-altered-adult⟧

Figure 4—figure supplement 3. Abnormal intrinsic physiology in parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) of the reticular thalamus in Kcnc1 -A421V/+ mice.⟦>zach claim=37d23efe-7e34-48aa-82ff-b7f3ef45751c: @{Figure 4—figure supplement 3. Abnormal intrinsic physiology in parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) of the reticular thalamus in Kcnc1 -A421V/+ mice.} rtn-neurons-impaired-excitability⟧

( A–B ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) (A, black ) and Kcnc1 -A421V/+ (B, green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA from their resting membrane potential without DC bias current (as in Figure 4 ).⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{( A–B ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) (A, black ) and Kcnc1 -A421V/+ (B, green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA from their resting membrane potential without DC bias current (as in Figure 4 ).} pv-in-ap-waveform-altered-downstroke-apd50⟧

( C ) Average frequency-current relationship for WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=32 cells, N = 12 mice) PV-INs with uncorrected resting membrane potential.⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{( C ) Average frequency-current relationship for WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=32 cells, N = 12 mice) PV-INs with uncorrected resting membrane potential.} pv-ins-impaired-maximal-firing — This is the same neocortical PV-IN frequency-current impairment the claim states, measured without correcting resting membrane potential.⟧

Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.} a421v-mice-die-before-122d⟧

Table 1. Membrane and action potential properties of WT and Kcnc1 -A421V/+ neurons at P16–21. AP, action potential; ADP, afterdepolarization; AHP, afterhyperpolarization; P, postnatal day; WT, wild type.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Table 1. Membrane and action potential properties of WT and Kcnc1 -A421V/+ neurons at P16–21. AP, action potential; ADP, afterdepolarization; AHP, afterhyperpolarization; P, postnatal day; WT, wild type.} excitatory-neurons-unaffected-juvenile⟧

Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=20,9) –72.3±1.3 –39.8±0.8 253±8 –183±9 52.1±1.8 0.40±0.02 142±13 93±9 –63.7±1.0 Kcnc1 -A421V/+ (N=36, 12) –67.4±1.3 –41.6±0.5 252±9 –138±8 57.1±1.6 0.54±0.03 170±21 145±19 –63.0±0.8 Statistical comparison *p=0.017 p=0.065 p=0.96 **p=0.0012 p=0.055 **p=0.0053 p=0.29 p=0.19 p=0.62 Layer IV exc. cells WT (N=23, 3) –66.7±0.7 –43.4±0.7 303±13 –68.6±3.9 79.8±1.3 1.06±0.05 155±15 34±6 –60.2±0.5 Kcnc1 -A421V/+ (N=22,3) –67.6±1.0 –41.8±0.7 286±15 –64.5±3.7 77.8±1.6 1.10±0.04 159±14 37±6 –60.8±0.6 Statistical comparison p=0.49 p=0.11 p=0.38 p=0.45 p=0.35 p=0.46 p=0.86 p=0.69 p=0.47 Layer V PV-INs WT (N=15, 3) –65.9±1.2 –38.6±1.0 309±22 –223±17 57.5±1.9 0.37±0.02 147±13 85±11 –65.8±1.0 Kcnc1 -A421V/+ (N=12, 3) –66.3±1.7 –40.4±1.0 278±26 –164±15 59.0±2.1 0.47±0.03 141±17 97±13 –65.7±1.2 Statistical comparison p=0.82 p=0.21 p=0.38 *p=0.016 p=0.60 *p=0.014 p=0.76 p=0.49 p=0.92 RTN WT (N=18, 4) –55.1±1.4 –39.2±0.8 227±15 –189±11 49.2±1.8 0.38±0.02 247±39 24±7 –64.5±0.5 Kcnc1 -A421V/+ (N=19, 5) –57.5±2.3 –38.1±1.0 195±12 –160±7 45.6±1.7 0.42±0.02 236±30 48±10 –63.7±0.8 Statistical comparison p=0.39 p=0.41 p=0.10 *p=0.034 p=0.15 p=0.092 p=0.82 p=0.0805 p=0.40 The number of asterisks was determined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Table 2. Membrane and AP properties of adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=20,9) –72.3±1.3 –39.8±0.8 253±8 –183±9 52.1±1.8 0.40±0.02 142±13 93±9 –63.7±1.0 Kcnc1 -A421V/+ (N=36, 12) –67.4±1.3 –41.6±0.5 252±9 –138±8 57.1±1.6 0.54±0.03 170±21 145±19 –63.0±0.8 Statistical comparison *p=0.017 p=0.065 p=0.96 **p=0.0012 p=0.055 **p=0.0053 p=0.29 p=0.19 p=0.62 Layer IV exc. cells WT (N=23, 3) –66.7±0.7 –43.4±0.7 303±13 –68.6±3.9 79.8±1.3 1.06±0.05 155±15 34±6 –60.2±0.5 Kcnc1 -A421V/+ (N=22,3) –67.6±1.0 –41.8±0.7 286±15 –64.5±3.7 77.8±1.6 1.10±0.04 159±14 37±6 –60.8±0.6 Statistical comparison p=0.49 p=0.11 p=0.38 p=0.45 p=0.35 p=0.46 p=0.86 p=0.69 p=0.47 Layer V PV-INs WT (N=15, 3) –65.9±1.2 –38.6±1.0 309±22 –223±17 57.5±1.9 0.37±0.02 147±13 85±11 –65.8±1.0 Kcnc1 -A421V/+ (N=12, 3) –66.3±1.7 –40.4±1.0 278±26 –164±15 59.0±2.1 0.47±0.03 141±17 97±13 –65.7±1.2 Statistical comparison p=0.82 p=0.21 p=0.38 *p=0.016 p=0.60 *p=0.014 p=0.76 p=0.49 p=0.92 RTN WT (N=18, 4) –55.1±1.4 –39.2±0.8 227±15 –189±11 49.2±1.8 0.38±0.02 247±39 24±7 –64.5±0.5 Kcnc1 -A421V/+ (N=19, 5) –57.5±2.3 –38.1±1.0 195±12 –160±7 45.6±1.7 0.42±0.02 236±30 48±10 –63.7±0.8 Statistical comparison p=0.39 p=0.41 p=0.10 *p=0.034 p=0.15 p=0.092 p=0.82 p=0.0805 p=0.40 The number of asterisks was determined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Table 2. Membrane and AP properties of adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.} pv-in-ap-waveform-altered-downstroke-apd50⟧

AT, action potential; PV-INs, parvalbumin-positive fast-spiking GABAergic inhibitory interneurons; P, postnatal day; WT, wild type.

Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=14,3) –66.1±1.4 –41.5±0.9 294±25 –248±21 51.1±2.0 0.31±0.02 141±11 91±10 –67.6±1.1 Kcnc1 -A421V/+ (N=17, 5) –68.3±2.3 –43.1±0.6 324±16 –164±18 67.8±2.3 0.58±0.07 173±25 106±23 –66.0±1.2 Statistical comparison p=0.45 p=0.16 p=0.29 **p=0.0051 ***p<0.001 **p=0.0026 p=0.28 p=0.87 p=0.34 Layer IV exc. cells WT (N=12,4) –68.6±0.9 –41.7±0.5 363±17 –83.2±5.1 83.4±0.9 0.89±0.04 110±10 88±10 –55.7±1.1 Kcnc1 -A421V/+ (N=12, 4) –67.2±0.5 –40.0±0.7 338±18 –78.6±4.7 81.0±1.7 0.92±0.03 131±10 58±8 –56.8±0.9 Statistical comparison p=0.19 p=0.057 p=0.33 p=0.52 p=0.24 p=0.69 p=0.14 *p=0.023 p=0.47 The number of asterisks was determined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. We sought to further extend these results by examining other cell populations also linked to epilepsy pathogenesis.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=14,3) –66.1±1.4 –41.5±0.9 294±25 –248±21 51.1±2.0 0.31±0.02 141±11 91±10 –67.6±1.1 Kcnc1 -A421V/+ (N=17, 5) –68.3±2.3 –43.1±0.6 324±16 –164±18 67.8±2.3 0.58±0.07 173±25 106±23 –66.0±1.2 Statistical comparison p=0.45 p=0.16 p=0.29 **p=0.0051 ***p<0.001 **p=0.0026 p=0.28 p=0.87 p=0.34 Layer IV exc. cells WT (N=12,4) –68.6±0.9 –41.7±0.5 363±17 –83.2±5.1 83.4±0.9 0.89±0.04 110±10 88±10 –55.7±1.1 Kcnc1 -A421V/+ (N=12, 4) –67.2±0.5 –40.0±0.7 338±18 –78.6±4.7 81.0±1.7 0.92±0.03 131±10 58±8 –56.8±0.9 Statistical comparison p=0.19 p=0.057 p=0.33 p=0.52 p=0.24 p=0.69 p=0.14 *p=0.023 p=0.47 The number of asterisks was determined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. We sought to further extend these results by examining other cell populations also linked to epilepsy pathogenesis.} pv-in-ap-waveform-altered-downstroke-apd50⟧

Recordings of neocortical layer V PV-INs from juvenile (P16–21) Kcnc1 -A421V/+ mice exhibited more subtle abnormalities compared to littermate WT control PV-INs, showing reduction in AP frequency only at the largest current injection magnitudes (***p<0.001 for interaction between genotype and current injection, Figure 4—figure supplement 2 ).⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Recordings of neocortical layer V PV-INs from juvenile (P16–21) Kcnc1 -A421V/+ mice exhibited more subtle abnormalities compared to littermate WT control PV-INs, showing reduction in AP frequency only at the largest current injection magnitudes (***p<0.001 for interaction between genotype and current injection, Figure 4—figure supplement 2 ).} a421v-mice-die-before-122d⟧

These results are consistent with previous reports that Kv3.1 comprises a relatively lower proportion of the overall Kv3 expression in deeper layer cortical PV-INs due to higher relative levels of Kv3.2 expression ( Chow et al., 1999 ).

We also examined PV-positive neurons in the reticular thalamic nucleus (RTN), which predominantly express Kv3.1 and Kv3.3 ( Porcello et al., 2002 ; Espinosa et al., 2008 ).

In response to hyperpolarizing current injections of various magnitudes, RTN neurons from Kcnc1 -A421V/+ mice (P16–21; N=19 cells, 5 mice) generated fewer rebound APs than their WT counterparts (n=16 cells, N=4 mice; Figure 4—figure supplement 3 ).⟦>zach claim=37d23efe-7e34-48aa-82ff-b7f3ef45751c: @{In response to hyperpolarizing current injections of various magnitudes, RTN neurons from Kcnc1 -A421V/+ mice (P16–21; N=19 cells, 5 mice) generated fewer rebound APs than their WT counterparts (n=16 cells, N=4 mice; Figure 4—figure supplement 3 ).} rtn-neurons-impaired-excitability⟧

As in neocortical PV-INs, the relationship between AP frequency and depolarizing current injection was attenuated in reticular thalamic cells from Kcnc1 -A421V/+ mice relative to WT counterparts (*p=0.0109; Figure 4—figure supplement 3 ).⟦>zach claim=37d23efe-7e34-48aa-82ff-b7f3ef45751c: @{As in neocortical PV-INs, the relationship between AP frequency and depolarizing current injection was attenuated in reticular thalamic cells from Kcnc1 -A421V/+ mice relative to WT counterparts (*p=0.0109; Figure 4—figure supplement 3 ).} rtn-neurons-impaired-excitability⟧

And as in neocortical PV-INs, the magnitude of the downstroke velocity was significantly reduced in Kcnc1 -A421V/+ RTN neurons, while other parameters did not reach significance at this time point ( Table 1 ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{And as in neocortical PV-INs, the magnitude of the downstroke velocity was significantly reduced in Kcnc1 -A421V/+ RTN neurons, while other parameters did not reach significance at this time point ( Table 1 ).} excitatory-neurons-unaffected-juvenile⟧

Thus, impairments in intrinsic physiology extend beyond neocortical PV-INs to Kv3.1-expressing cells in other brain regions, but with cell populations known to express Kv3.2 or Kv3.3 showing more subtle impairment than PV-INs in superficial neocortical layers.

Normal physiological function in excitatory neurons from Kcnc1 -A421V/+ mice We next investigated the voltage-gated K + channel function and intrinsic excitability in layer IV excitatory cells from both WT and Kcnc1 -A421V/+ mice at P16–21 ( Figure 5 ) and P32–42 ( Figure 5—figure supplement 1 ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Normal physiological function in excitatory neurons from Kcnc1 -A421V/+ mice We next investigated the voltage-gated K + channel function and intrinsic excitability in layer IV excitatory cells from both WT and Kcnc1 -A421V/+ mice at P16–21 ( Figure 5 ) and P32–42 ( Figure 5—figure supplement 1 ).} excitatory-neurons-unaffected-juvenile⟧

While voltage-gated K + channel currents in excitatory neurons were of significantly lower magnitude compared to that observed in PV-INs, there were no genotype differences in K + currents between excitatory cells from WT and Kcnc1 -A421V/+ mice, consistent with a lack of Kv3.1 expression in excitatory cells ( Figure 5B–D ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{While voltage-gated K + channel currents in excitatory neurons were of significantly lower magnitude compared to that observed in PV-INs, there were no genotype differences in K + currents between excitatory cells from WT and Kcnc1 -A421V/+ mice, consistent with a lack of Kv3.1 expression in excitatory cells ( Figure 5B–D ).} excitatory-neurons-unaffected-juvenile⟧

Neither voltage-dependent current density ( Figure 5D ), peak current density ( Figure 5E ), voltage-dependent activation ( Figure 5F ), nor the voltage-dependent rate of activation ( Figure 5G ) was altered in excitatory neurons of Kcnc1 -A421V/+ mice relative to WT controls.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Neither voltage-dependent current density ( Figure 5D ), peak current density ( Figure 5E ), voltage-dependent activation ( Figure 5F ), nor the voltage-dependent rate of activation ( Figure 5G ) was altered in excitatory neurons of Kcnc1 -A421V/+ mice relative to WT controls.} excitatory-neurons-unaffected-juvenile⟧

We also recorded these cells in current clamp to characterize intrinsic excitability ( Figure 5H–K ).⟦>zach claim=no-assertion: @{We also recorded these cells in current clamp to characterize intrinsic excitability ( Figure 5H–K ).} Narration of which recording mode was used, reporting no result.⟧

Across a range of depolarizing current injection magnitudes, we did not detect any differences in steady-state AP frequency in excitatory neurons between WT and Kcnc1 -A421V/+ mice ( Figure 5I–K ).

We also did not detect any statistically significant alterations in the passive membrane properties or properties of single APs between WT and Kcnc1 -A421V/+ mice ( Table 1 ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{We also did not detect any statistically significant alterations in the passive membrane properties or properties of single APs between WT and Kcnc1 -A421V/+ mice ( Table 1 ).} excitatory-neurons-unaffected-juvenile⟧

At the adult time point (P32–42), we similarly did not observe spiking differences ( Figure 5—figure supplement 1 ; Table 2 ).⟦>zach claim=3df55d45-b8a0-42c6-9807-0323c08e8815: @{At the adult time point (P32–42), we similarly did not observe spiking differences ( Figure 5—figure supplement 1 ; Table 2 ).} excitatory-neurons-unaffected-adult⟧

Overall, these data suggest that intrinsic excitability of neocortical excitatory neurons is not altered in juvenile or adult Kcnc1 -A421V/+ mice (either directly or via a secondary network effect), while impaired intrinsic excitability of PV-positive neurons in the neocortex and reticular thalamus is most likely a direct result of cell-autonomous reduction in Kv3.1 current density.

Figure 5. Unaltered potassium currents and physiological function of excitatory neurons in postnatal day (P)16–21 Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Figure 5. Unaltered potassium currents and physiological function of excitatory neurons in postnatal day (P)16–21 Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile⟧

( A ) Representative image of a neocortical excitatory cell being recorded in the outside-out nucleated macropatch configuration.⟦>zach claim=no-assertion: @{( A ) Representative image of a neocortical excitatory cell being recorded in the outside-out nucleated macropatch configuration.} A representative image of the recording configuration, asserting nothing about genotype.⟧

( B–C ) Example family of traces of voltage-gated K + channel currents from an excitatory cell from wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( B–C ) Example family of traces of voltage-gated K + channel currents from an excitatory cell from wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) mice.} excitatory-neurons-unaffected-juvenile⟧

( D ) Average voltage-gated K + channel current density for WT (n=8 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=10, N=3 mice) relative to membrane potential.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( D ) Average voltage-gated K + channel current density for WT (n=8 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=10, N=3 mice) relative to membrane potential.} excitatory-neurons-unaffected-juvenile⟧

(E) Peak voltage-gated K + channel current density in WT and Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{(E) Peak voltage-gated K + channel current density in WT and Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile⟧

( F ) Normalized voltage-dependent activation curves for WT and Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( F ) Normalized voltage-dependent activation curves for WT and Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile⟧

( G ) Average voltage-dependent activation time constant for the voltage-gated K + channel currents.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( G ) Average voltage-dependent activation time constant for the voltage-gated K + channel currents.} excitatory-neurons-unaffected-juvenile⟧

( H ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical excitatory cell recorded in the whole-cell configuration to characterize intrinsic excitability.⟦>zach claim=no-assertion: @{( H ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical excitatory cell recorded in the whole-cell configuration to characterize intrinsic excitability.} A representative image of the recorded cell, asserting nothing about genotype.⟧

( I–J ) Representative example traces showing excitatory cell action potential (AP) generation in WT ( I , black ) and Kcnc1 -A421V/+ ( J , green ) in response to depolarizing current injections.

( K ) Average relationship between excitatory cell AP frequency in response to a range of current injections for WT (N=23 cells, N=3 mice) and Kcnc1 -A421V/+ (n=22, N=3 mice).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( K ) Average relationship between excitatory cell AP frequency in response to a range of current injections for WT (N=23 cells, N=3 mice) and Kcnc1 -A421V/+ (n=22, N=3 mice).} excitatory-neurons-unaffected-juvenile — This panel displays the juvenile excitatory-cell frequency-current relationship the claim reports as unchanged.⟧

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA or unpaired t-test.

Figure 5—figure supplement 1. Intrinsic excitability is unchanged in excitatory cells from postnatal day (P)32 to P42 Kcnc1 -A421V/+ mice.⟦>zach claim=3df55d45-b8a0-42c6-9807-0323c08e8815: @{Figure 5—figure supplement 1. Intrinsic excitability is unchanged in excitatory cells from postnatal day (P)32 to P42 Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-adult⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a recorded layer IV excitatory cell.⟦>zach claim=no-assertion: @{( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a recorded layer IV excitatory cell.} A representative image of the recorded cell, asserting nothing about genotype.⟧

( B–C ) Representative example traces of evoked action potentials (APs) in response to depolarizing current injections in cortical layer IV excitatory neurons from wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) mice.⟦>zach claim=no-assertion: @{( B–C ) Representative example traces of evoked action potentials (APs) in response to depolarizing current injections in cortical layer IV excitatory neurons from wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) mice.} Example traces of evoked action potentials, with no result stated.⟧

( D ) Average frequency-current relationship for WT (black; n=12, N=4) and Kcnc1 -A421V/+ (n=12, N=4) mice.⟦>zach claim=3df55d45-b8a0-42c6-9807-0323c08e8815: @{( D ) Average frequency-current relationship for WT (black; n=12, N=4) and Kcnc1 -A421V/+ (n=12, N=4) mice.} excitatory-neurons-unaffected-adult — This panel displays the adult (P32-42) excitatory-cell frequency-current relationship the claim reports as showing no genotype difference.⟧

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA.

PV-IN and excitatory cell synaptic neurotransmission is functionally intact in juvenile Kcnc1 -A421V/+ mice Within fast-spiking cells, Kv3 channels are expressed in specific subcellular compartments and are functionally involved not only in AP generation, but also in AP propagation along the axon, and in inhibitory neurotransmission at the synaptic terminal via regulation of synaptic AP waveform ( Goldberg et al., 2005 ; Rowan et al., 2014 ; Rowan et al., 2016 ; Rowan and Christie, 2017 ).

Additionally, prior work has shown that blocking presynaptic Kv3 current leads to an increase in the efficacy of synaptic transmission, albeit with enhanced short-term synaptic depression ( Ishikawa et al., 2003 ; Brooke et al., 2004 ; Goldberg et al., 2005 ).

For that reason, we sought to determine the impact of the A421V variant on PV-IN-mediated inhibitory synaptic neurotransmission in juvenile (P16–21) WT vs. Kcnc1 -A421V/+ mice ( Figure 6 ).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{For that reason, we sought to determine the impact of the A421V variant on PV-IN-mediated inhibitory synaptic neurotransmission in juvenile (P16–21) WT vs. Kcnc1 -A421V/+ mice ( Figure 6 ).} pv-in-inhibitory-synapse-intact-juvenile⟧

We collected simultaneous whole-cell patch-clamp electrophysiology recordings from one neocortical layer II-IV PV-IN and one nearby (<100 μm inter-soma distance) excitatory neuron, of which 21 of 64 (32.8%) WT neuron pairs and 15 of 43 (34.9%) Kcnc1 -A421V/+ neuron pairs exhibited unitary inhibitory postsynaptic currents (uIPSCs) in the excitatory cell in response to AP generation in the PV-IN at 5, 10, 20, 40, 80, and 120 Hz ( Figure 6A–E ).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{We collected simultaneous whole-cell patch-clamp electrophysiology recordings from one neocortical layer II-IV PV-IN and one nearby (<100 μm inter-soma distance) excitatory neuron, of which 21 of 64 (32.8%) WT neuron pairs and 15 of 43 (34.9%) Kcnc1 -A421V/+ neuron pairs exhibited unitary inhibitory postsynaptic currents (uIPSCs) in the excitatory cell in response to AP generation in the PV-IN at 5, 10, 20, 40, 80, and 120 Hz ( Figure 6A–E ).} pv-in-inhibitory-synapse-intact-juvenile — The claim reports exactly these juvenile paired-recording connection rates, 21 of 64 WT versus 15 of 43 mutant pairs.⟧

Rates of failure of the first five APs (AP is successfully initiated in the PV-IN, but no uIPSC is observed in the excitatory cell) were not different in WT vs. Kcnc1 -A421V/+ mice ( Figure 6F ).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{Rates of failure of the first five APs (AP is successfully initiated in the PV-IN, but no uIPSC is observed in the excitatory cell) were not different in WT vs. Kcnc1 -A421V/+ mice ( Figure 6F ).} pv-in-inhibitory-synapse-intact-juvenile⟧

The magnitudes of the first five uIPSCs at various stimulation frequencies were also not significantly different between the two genotypes ( Figure 6G–I ).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{The magnitudes of the first five uIPSCs at various stimulation frequencies were also not significantly different between the two genotypes ( Figure 6G–I ).} pv-in-inhibitory-synapse-intact-juvenile⟧

The paired-pulse ratios, either uIPSC 2 /uIPSC 1 or uIPSC Last /uIPSC 1 , were not different between WT and Kcnc1 -A421V/+ ( Figure 6J and K ).

Finally, we did not detect a significant difference in synaptic latency of the uIPSC between WT and Kcnc1 -A421V/+ neuron pairs ( Figure 6L ).

These data suggest that, despite expression of Kv3.1 in the axon and synaptic terminal in WT mice, neocortical PV-IN-mediated inhibitory synaptic neurotransmission remains intact in Kcnc1 -A421V/+ mice at this juvenile developmental time point.

Yet, inhibitory transmission will be secondarily impaired in Kcnc1- A421V/+ mice secondary to the abnormal excitability and impaired spike generation of PV-INs.

Figure 6. Juvenile (P16–21) Kcnc1 -A421V/+ mice exhibit normal parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated inhibitory synaptic neurotransmission.⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{Figure 6. Juvenile (P16–21) Kcnc1 -A421V/+ mice exhibit normal parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated inhibitory synaptic neurotransmission.} pv-in-inhibitory-synapse-intact-juvenile⟧

( A ) Representative images showing simultaneous whole-cell patch-clamp recordings of cortical PV-IN and nearby excitatory cell ( left , ×10 magnification; right, ×40 magnification).⟦>zach claim=no-assertion: @{( A ) Representative images showing simultaneous whole-cell patch-clamp recordings of cortical PV-IN and nearby excitatory cell ( left , ×10 magnification; right, ×40 magnification).} A representative image of the paired-recording configuration, asserting nothing about genotype.⟧

( B ) Example traces of a PV-IN and excitatory cell pair in which generation of action potentials (APs) in the PV-IN (bottom trace) is sufficient to induce clear unitary inhibitory postsynaptic potentials (uIPSPs) in the excitatory cell (arrowhead, top trace).⟦>zach claim=no-assertion: @{( B ) Example traces of a PV-IN and excitatory cell pair in which generation of action potentials (APs) in the PV-IN (bottom trace) is sufficient to induce clear unitary inhibitory postsynaptic potentials (uIPSPs) in the excitatory cell (arrowhead, top trace).} An example trace with its arrowhead annotation, not a stated finding.⟧

The inset shows an example view of the individual uIPSPs corresponding to each AP in the PV-IN.

( C–D ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both wild-type (WT) ( C ) and Kcnc1 -A421V/+ pairs of synaptically connected neurons. 10 APs were generated in the PV-IN (top trace) at 40 Hz and the uIPSCs are shown below.⟦>zach claim=no-assertion: @{( C–D ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both wild-type (WT) ( C ) and Kcnc1 -A421V/+ pairs of synaptically connected neurons. 10 APs were generated in the PV-IN (top trace) at 40 Hz and the uIPSCs are shown below.} Example traces and the stimulation protocol used to generate them, with no result stated.⟧

The black and green traces are the averages of numerous individual sweeps shown in gray.

( E ) Probability of synaptic connection between PV-IN and excitatory cell in WT (n=21 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=5 of 43 pairs from N=11 mice).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{( E ) Probability of synaptic connection between PV-IN and excitatory cell in WT (n=21 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=5 of 43 pairs from N=11 mice).} pv-in-inhibitory-synapse-intact-juvenile⟧

( F ) Average failure probability relative to presynaptic stimulation frequency in WT and Kcnc1 -A421V/+ neuron pairs.⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{( F ) Average failure probability relative to presynaptic stimulation frequency in WT and Kcnc1 -A421V/+ neuron pairs.} pv-in-inhibitory-synapse-intact-juvenile⟧

( G–I ) Average uIPSC magnitude for the first five APs in WT and Kcnc1 -A421V/+ at 20 Hz ( G ), 40 Hz ( H ), and 80 Hz ( I ) stimulus frequencies.⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{( G–I ) Average uIPSC magnitude for the first five APs in WT and Kcnc1 -A421V/+ at 20 Hz ( G ), 40 Hz ( H ), and 80 Hz ( I ) stimulus frequencies.} pv-in-inhibitory-synapse-intact-juvenile⟧

( J–K ) Paired-pulse ratios for both WT and Kcnc1 -A421V/+ mice relative to stimulus frequency.

The ratio of second uIPSC to the first is provided in J , while K displays the average ratio of the last uIPSC to the first.

(L) Average latency from peak of presynaptic AP to peak of the uIPSC in WT and Kcnc1 -A421V/+ mice.

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA or unpaired t-test.

Figure 6—figure supplement 1. Excitatory neuron to parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) unitary excitatory synaptic neurotransmission is unaltered in juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Figure 6—figure supplement 1. Excitatory neuron to parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) unitary excitatory synaptic neurotransmission is unaltered in juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile — The claim states that synaptic transmission involving juvenile excitatory neurons is unaltered, which is this supplement's finding for excitatory-to-PV-IN transmission.⟧

( A ) Example traces of simultaneous recordings of a synaptically connected PV-IN and excitatory cell neuron pair in which generation of action potentials (APs) in the excitatory cell leads to unitary excitatory postsynaptic potentials (uEPSPs) in the PV-IN.⟦>zach claim=no-assertion: @{( A ) Example traces of simultaneous recordings of a synaptically connected PV-IN and excitatory cell neuron pair in which generation of action potentials (APs) in the excitatory cell leads to unitary excitatory postsynaptic potentials (uEPSPs) in the PV-IN.} An example trace of a connected pair, with no result stated.⟧

The inset shows an expanded view of the uEPSPs in the PV-IN generated from each AP in the excitatory cell.

( B–C ) Representative example traces of unitary excitatory postsynaptic currents (uEPSCs) generated in wild-type (WT) ( B ) and Kcnc1 -A421V/+ pairs of excitatory cells (top trace) and nearby PV-INs (bottom trace).⟦>zach claim=no-assertion: @{( B–C ) Representative example traces of unitary excitatory postsynaptic currents (uEPSCs) generated in wild-type (WT) ( B ) and Kcnc1 -A421V/+ pairs of excitatory cells (top trace) and nearby PV-INs (bottom trace).} Example traces with their colour coding, not a stated finding.⟧

In response to a 20 Hz train of five APs generated in the excitatory cells, unitary inhibitory postsynaptic currents (uIPSCs) are recorded in the PV-INs (shown in gray), with the average of numerous sweeps shown for wild-type (WT) (black) or for Kcnc1 -A421V/+ (green).

( D ) Connection probability between the excitatory cell and the PV-IN for WT (n=9 of 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=6 of 43 pairs from N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) Connection probability between the excitatory cell and the PV-IN for WT (n=9 of 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=6 of 43 pairs from N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

( E ) Average failure rate for WT and Kcnc1 -A421V/+ neuron pairs relative to presynaptic stimulation frequency.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( E ) Average failure rate for WT and Kcnc1 -A421V/+ neuron pairs relative to presynaptic stimulation frequency.} excitatory-neurons-unaffected-juvenile — Failure rate of excitatory-to-PV-IN transmission is part of the unaltered juvenile synaptic transmission the claim reports.⟧

( F ) Average uEPSC magnitude in response to a train of five APs generated at 20 Hz in WT and Kcnc1 -A421V/+ neuron pairs.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( F ) Average uEPSC magnitude in response to a train of five APs generated at 20 Hz in WT and Kcnc1 -A421V/+ neuron pairs.} excitatory-neurons-unaffected-juvenile — uEPSC magnitude is part of the unaltered juvenile excitatory synaptic transmission the claim reports.⟧

( G ) Paired-pulse ratio of the second uEPSC to the first uEPSC in WT and Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( G ) Paired-pulse ratio of the second uEPSC to the first uEPSC in WT and Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile — Paired-pulse ratio of the uEPSC is part of the unaltered juvenile excitatory synaptic transmission the claim reports.⟧

(H) Average synaptic latency between peak of AP to peak of uEPSC in both WT and Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{(H) Average synaptic latency between peak of AP to peak of uEPSC in both WT and Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile — uEPSC synaptic latency is part of the unaltered juvenile excitatory synaptic transmission the claim reports.⟧

In 9 of 61 neuron (14.8%) pairs for WT mice and 6 of 43 pairs (14.0%) for Kcnc1 -A421V/+ mice at P16–21, we observed unitary excitatory synaptic currents (uEPSCs) from the excitatory neuron onto the PV-IN, which allowed us to investigate whether there were abnormalities in excitatory neurotransmission ( Figure 6—figure supplement 1A–D ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{In 9 of 61 neuron (14.8%) pairs for WT mice and 6 of 43 pairs (14.0%) for Kcnc1 -A421V/+ mice at P16–21, we observed unitary excitatory synaptic currents (uEPSCs) from the excitatory neuron onto the PV-IN, which allowed us to investigate whether there were abnormalities in excitatory neurotransmission ( Figure 6—figure supplement 1A–D ).} excitatory-neurons-unaffected-juvenile — The near-identical excitatory-to-PV-IN connection rates in the two genotypes are part of the unaltered juvenile excitatory synaptic transmission the claim reports.⟧

The frequency-dependent rate of failure showed no significant effect for genotype ( Figure 6—figure supplement 1E ).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{The frequency-dependent rate of failure showed no significant effect for genotype ( Figure 6—figure supplement 1E ).} excitatory-neurons-unaffected-juvenile — The null genotype effect on failure rate is part of the unaltered juvenile excitatory synaptic transmission the claim reports.⟧

Lastly, the magnitude ( Figure 6—figure supplement 1F ), paired-pulse ratio ( Figure 6—figure supplement 1G ), and the latency ( Figure 6—figure supplement 1H ) of the uEPSCs were not significantly different between WT and Kcnc1 -A421V/+ neuron pairs, indicating that excitatory neuron synaptic transmission onto PV-INs is not altered in juvenile Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Lastly, the magnitude ( Figure 6—figure supplement 1F ), paired-pulse ratio ( Figure 6—figure supplement 1G ), and the latency ( Figure 6—figure supplement 1H ) of the uEPSCs were not significantly different between WT and Kcnc1 -A421V/+ neuron pairs, indicating that excitatory neuron synaptic transmission onto PV-INs is not altered in juvenile Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile — This is the summary statement of the claim's own finding: excitatory transmission onto PV-INs is unaltered in juvenile mutants.⟧

PV-IN synaptic neurotransmission is altered in adult Kcnc1 -A421V/+ mice As Kv3.1 exhibits developmentally regulated expression, we next assessed PV-IN-mediated inhibitory neurotransmission in young adult (P32–42) Kcnc1 -A421V/+ mice relative to WT controls ( Figure 7 ).⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{PV-IN synaptic neurotransmission is altered in adult Kcnc1 -A421V/+ mice As Kv3.1 exhibits developmentally regulated expression, we next assessed PV-IN-mediated inhibitory neurotransmission in young adult (P32–42) Kcnc1 -A421V/+ mice relative to WT controls ( Figure 7 ).} pv-in-inhibitory-synapse-altered-adult⟧

As in the younger mice, we recorded pairs of cortical PV-IN and excitatory neurons that were synaptically connected – 14 of 36 pairs (38.9%) in WT mice (N = 8 mice) and 13 of 36 pairs (36.1%) in Kcnc1 -A421V/+ mice (N=8 mice; Figure 7A–D ).⟦>zach claim=gap: @{As in the younger mice, we recorded pairs of cortical PV-IN and excitatory neurons that were synaptically connected – 14 of 36 pairs (38.9%) in WT mice (N = 8 mice) and 13 of 36 pairs (36.1%) in Kcnc1 -A421V/+ mice (N=8 mice; Figure 7A–D ).} No claim records the adult PV-IN to excitatory connection probability, 14 of 36 WT versus 13 of 36 mutant pairs; the adult synapse claim covers only uIPSC magnitude and paired-pulse ratio.⟧

We did not detect differences in failure rates between WT and Kcnc1 -A421V/+ mice over a range of stimulation frequencies ( Figure 7E ).⟦>zach claim=gap: @{We did not detect differences in failure rates between WT and Kcnc1 -A421V/+ mice over a range of stimulation frequencies ( Figure 7E ).} No claim records that release failure rates are unchanged in adult mutants; the adult synapse claim states only the increased uIPSC magnitude and reduced paired-pulse ratio.⟧

Notably, the average magnitude of the first five uIPSCs was significantly increased in Kcnc1 -A421V/+ relative to WT recordings (**p<0.01 at 20 Hz, *p<0.05 at 40 Hz, and *p<0.05 at 80 Hz; Figure 7F–H ).⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Notably, the average magnitude of the first five uIPSCs was significantly increased in Kcnc1 -A421V/+ relative to WT recordings (**p<0.01 at 20 Hz, *p<0.05 at 40 Hz, and *p<0.05 at 80 Hz; Figure 7F–H ).} pv-in-inhibitory-synapse-altered-adult⟧

Additionally, we observed a reduced paired-pulse ratio for the second uIPSC relative to the first uIPSC in Kcnc1 -A421V/+ neuron pairs from young adult mice across a range of stimulation frequencies (*p<0.05; Figure 7I ), but found no genotype effect in the average ratio between the last uIPSC to the first uIPSC ( Figure 7J ).⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Additionally, we observed a reduced paired-pulse ratio for the second uIPSC relative to the first uIPSC in Kcnc1 -A421V/+ neuron pairs from young adult mice across a range of stimulation frequencies (*p<0.05; Figure 7I ), but found no genotype effect in the average ratio between the last uIPSC to the first uIPSC ( Figure 7J ).} pv-in-inhibitory-synapse-altered-adult⟧

The uIPSC latency measured from AP peak to onset of the synaptic event was not significantly different between WT and Kcnc1 -A421V/+ neuron pairs ( Figure 7K ).

Overall, these complex results align with prior work, showing that Kv3.1 is an important regulator of synaptic neurotransmission in PV-INs and suggests that synaptic dysfunction may contribute to the pathogenesis of epilepsy in Kcnc1 -A421V/+ mice in a developmentally determined manner.

Figure 7. Adult (P32–42) Kcnc1 -A421V/+ mice exhibit altered parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated synaptic neurotransmission.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Figure 7. Adult (P32–42) Kcnc1 -A421V/+ mice exhibit altered parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated synaptic neurotransmission.} pv-in-inhibitory-synapse-altered-adult⟧

( A ) Example presynaptic cortical PV-IN and postsynaptic excitatory neuron.⟦>zach claim=no-assertion: @{( A ) Example presynaptic cortical PV-IN and postsynaptic excitatory neuron.} An example pair with an annotated inset, with no result stated.⟧

Arrowhead and inset display the unitary inhibitory postsynaptic potentials (uIPSPs) induced in the postsynaptic cell when the PV-IN generates action potentials (APs).

( B–C ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both adult wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) pairs of synaptically connected neurons. 10 APs were generated in the PV-IN at 40 Hz, and the evoked uIPSCs are displayed in the trace below where the black and green traces are the averages of numerous individual sweeps shown in gray.⟦>zach claim=no-assertion: @{( B–C ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both adult wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) pairs of synaptically connected neurons. 10 APs were generated in the PV-IN at 40 Hz, and the evoked uIPSCs are displayed in the trace below where the black and green traces are the averages of numerous individual sweeps shown in gray.} Example traces and the convention for averaged versus individual sweeps, not a stated finding.⟧

( D ) Connection probability between WT (14 of 36, N=8 mice) and Kcnc1 -A421V/+ (13 of 36, N=8 mice) pairs PV-INs and nearby excitatory cells.⟦>zach claim=gap: @{( D ) Connection probability between WT (14 of 36, N=8 mice) and Kcnc1 -A421V/+ (13 of 36, N=8 mice) pairs PV-INs and nearby excitatory cells.} This panel carries the adult PV-IN to excitatory connection probability, which no claim in the tree records.⟧

( E ) Average frequency-dependent rate of failure for the first five APs in adult WT and Kcnc1 -A421V/+ neuron pairs.⟦>zach claim=gap: @{( E ) Average frequency-dependent rate of failure for the first five APs in adult WT and Kcnc1 -A421V/+ neuron pairs.} This panel carries the adult frequency-dependent failure rates, which no claim in the tree records.⟧

(F–H) Average uIPSC magnitude of the first five APs for adult WT and Kcnc1 -A421V/+ at 20 Hz ( F ), 40 Hz ( G ), and 80 Hz ( H ).⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{(F–H) Average uIPSC magnitude of the first five APs for adult WT and Kcnc1 -A421V/+ at 20 Hz ( F ), 40 Hz ( G ), and 80 Hz ( H ).} pv-in-inhibitory-synapse-altered-adult⟧

(I–J) Paired-pulse ratios for WT and Kcnc1 -A421V/+ neuron pairs (uIPSC 2 /uIPSC 1 provided in I , uIPSC last /uIPSC first provided in J ) relative to stimulus frequency.

( K ) Average latency from AP peak to onset of the uIPSC in WT and Kcnc1 -A421V/+ mice.

Data are shown as mean ± SEM or individual values, and significance (*p<0.05, **p<0.01) was determined using unpaired t-test or repeated-measures two-way ANOVA.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM or individual values, and significance (*p<0.05, **p<0.01) was determined using unpaired t-test or repeated-measures two-way ANOVA.} pv-in-inhibitory-synapse-altered-adult⟧

Two-photon in vivo calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice We then utilized two-photon (2P) calcium imaging to investigate neural activity in neocortical circuits in both WT and Kcnc1 -A421V/+ mice (>P50) in layer II/III of primary somatosensory cortex in vivo ( Figure 8A ).⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{Two-photon in vivo calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice We then utilized two-photon (2P) calcium imaging to investigate neural activity in neocortical circuits in both WT and Kcnc1 -A421V/+ mice (>P50) in layer II/III of primary somatosensory cortex in vivo ( Figure 8A ).} in-vivo-hypersynchronous-discharges-mutant-only — The claim reports the paroxysmal hypersynchronous discharges found by two-photon imaging in mice over P50 that this section announces.⟧

We observed striking instances of paroxysmal hypersynchronous discharges in the neuropil signal of seven of seven Kcnc1 -A421V/+ mice examined, but never in WT mice ( Figure 8B–D ).⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{We observed striking instances of paroxysmal hypersynchronous discharges in the neuropil signal of seven of seven Kcnc1 -A421V/+ mice examined, but never in WT mice ( Figure 8B–D ).} in-vivo-hypersynchronous-discharges-mutant-only⟧

During each hypersynchronous discharge, the mouse was stationary and non-ambulatory but displayed a brief diffuse twitch involving the facial musculature and bilateral limbs ( Video 1 ).

Taken together, these in vivo imaging results indicate that Kcnc1 -A421V/+ mice exhibit paroxysmal synchronous discharges that may correlate with seizures, potentially myoclonic seizures.

Figure 8. In vivo two-photon (2P) calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice.⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{Figure 8. In vivo two-photon (2P) calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice.} in-vivo-hypersynchronous-discharges-mutant-only⟧

( A ) Experimental setup for in vivo 2P calcium imaging with representative calcium transients from cells expressing AAV-hSyn-GCaMP8m and mean dF/F 0 of the whole field of view (FOV) aligned to locomotion speed.⟦>zach claim=no-assertion: @{( A ) Experimental setup for in vivo 2P calcium imaging with representative calcium transients from cells expressing AAV-hSyn-GCaMP8m and mean dF/F 0 of the whole field of view (FOV) aligned to locomotion speed.} A description of the imaging setup and what the representative traces show, not a finding.⟧

( B ) Representative 2P field of view during a hypersynchronous discharge in a Kcnc1 -A421V/+ mouse.⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{( B ) Representative 2P field of view during a hypersynchronous discharge in a Kcnc1 -A421V/+ mouse.} in-vivo-hypersynchronous-discharges-mutant-only⟧

( C ) Mean dF/F of the field of view (top), calcium transients of individual somata (middle), and locomotion speed (bottom) during a paroxysmal discharge in the Kcnc1 -A421V/+ mouse relative to typical baseline activity shown in a wild-type (WT) mouse.⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{( C ) Mean dF/F of the field of view (top), calcium transients of individual somata (middle), and locomotion speed (bottom) during a paroxysmal discharge in the Kcnc1 -A421V/+ mouse relative to typical baseline activity shown in a wild-type (WT) mouse.} in-vivo-hypersynchronous-discharges-mutant-only⟧

Note that, in contrast to epochs of low-amplitude synchronization in WT associated with transition from quiet wakefulness to locomotion, there is no locomotion during the larger-amplitude hypersynchronous discharges identified in Kcnc1 -A421V/+ mice.

( D ) Frequency of paroxysmal discharges in each mouse (N=5 WT, N = 7 Kcnc1 -A421V/+ mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) Frequency of paroxysmal discharges in each mouse (N=5 WT, N = 7 Kcnc1 -A421V/+ mice).} a421v-kv31-membrane-trafficking-impaired⟧

( E ) Experimental design for in vivo 2P calcium imaging of somata positive (PV+) and negative (PV–) for parvalbumin.⟦>zach claim=no-assertion: @{( E ) Experimental design for in vivo 2P calcium imaging of somata positive (PV+) and negative (PV–) for parvalbumin.} A description of the experimental design for PV+/PV- imaging, not a finding.⟧

( F ) Example calcium transients of PV+ (bottom) and PV– (top) somata aligned to locomotion speed in a WT (left) and Kcnc1 -A421V mouse (right).⟦>zach claim=no-assertion: @{( F ) Example calcium transients of PV+ (bottom) and PV– (top) somata aligned to locomotion speed in a WT (left) and Kcnc1 -A421V mouse (right).} Example transients with their layout convention, not a stated finding.⟧

( G–H ) Transients per minute during quiet rest in ( G ) PV– (WT, N=n=885 cells, 4 mice, mean = 1.17; Kcnc1 -A421V, n=1041 cells, N=3 mice, mean = 1.63) and ( H ) PV+ cells (WT, N=4 mice, n=110 cells, mean = 0.94; Kcnc1 -A421V, N=3 mice, n=100 cells, mean = 1.50).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{( G–H ) Transients per minute during quiet rest in ( G ) PV– (WT, N=n=885 cells, 4 mice, mean = 1.17; Kcnc1 -A421V, n=1041 cells, N=3 mice, mean = 1.63) and ( H ) PV+ cells (WT, N=4 mice, n=110 cells, mean = 0.94; Kcnc1 -A421V, N=3 mice, n=100 cells, mean = 1.50).} in-vivo-pv-minus-transient-frequency-increased⟧

( I–J ) Mean peak height in ( I ) PV– (WT, mean = 0.46; Kcnc1 -A421V, mean = 0.41) and ( J ) PV+ cells (WT, mean = 0.48; Kcnc1 -A421V , mean = 0.40).

Data points are shaded by mouse identity.

Statistical comparisons were performed using mixed-effects modeling.

Figure 8—figure supplement 1. Analysis of in vivo two-photon calcium imaging data.⟦>zach claim=no-assertion: @{Figure 8—figure supplement 1. Analysis of in vivo two-photon calcium imaging data.} A bare supplement title naming the analysis, with no finding asserted.⟧

( A ) PV– (wild-type [WT], n=847 cells, N=4 mice; Kcnc1 -A421V, n=901 cells, N=3 mice) and ( B ) PV+ cells (WT, n=65 cells; Kcnc1 -A421V, n=60 cells).⟦>zach claim=no-assertion: @{( A ) PV– (wild-type [WT], n=847 cells, N=4 mice; Kcnc1 -A421V, n=901 cells, N=3 mice) and ( B ) PV+ cells (WT, n=65 cells; Kcnc1 -A421V, n=60 cells).} A panel label giving only the cell and mouse counts for each group.⟧

( C–D ) Transients per minute during running in ( C ) PV– (WT, n=885 cells, N=4 mice; Kcnc1 -A421V, n=1041 cells, N=3 mice) and ( D ) PV+ cells (WT, n=110 cells, N=4 mice; Kcnc1 -A421V, n=100 cells, N=3 mice).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{( C–D ) Transients per minute during running in ( C ) PV– (WT, n=885 cells, N=4 mice; Kcnc1 -A421V, n=1041 cells, N=3 mice) and ( D ) PV+ cells (WT, n=110 cells, N=4 mice; Kcnc1 -A421V, n=100 cells, N=3 mice).} in-vivo-pv-minus-transient-frequency-increased⟧

( E–F ) Mean transient height in ( E ) PV– and ( F ) PV+ cells.⟦>zach claim=gap: @{( E–F ) Mean transient height in ( E ) PV– and ( F ) PV+ cells.} No claim records the transient heights measured during running epochs; the in vivo claims cover only the quiet-rest state.⟧

Error bars indicate standard error of the mean, and values next to data points are the mean by genotype.

Data points are shaded by mouse identity.

Statistical comparisons were performed using mixed-effects modeling.

( G–H ) Boxplot of percent of active ( G ) PV– and ( H ) PV+ cells for each mouse.⟦>zach claim=gap: @{( G–H ) Boxplot of percent of active ( G ) PV– and ( H ) PV+ cells for each mouse.} No claim records the percentage of active PV- and PV+ cells per mouse that this panel carries.⟧

Video 1. Example in vivo two-photon (2P) calcium imaging of synchronous discharge in a Kcnc1- A421V/+ mouse.

Because the occurrence of large-amplitude hypersynchronous discharges in the neuropil contaminated the somatic signal in these recordings, we performed in vivo 2P imaging in a separate cohort of WT and Kcnc1 -A421V/+ mice that were co-injected with a pan-neuronal soma-tagged GCaMP8m and an S5E2 enhancer-driven tdTomato to identify PV+ interneurons.

Interestingly, in this cohort, we did not detect hypersynchronous discharges in Kcnc1 -A421V/+ mice, suggesting that this phenomenon is primarily localized to the neuropil.

Analysis of PV– and PV+ cell activity revealed that, during epochs of quiet rest (when the mouse was stationary on the treadmill), PV– cells on average displayed more frequent calcium transients, whereas there was no change in the frequency of PV+ cell transients ( Figure 8G–H ).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{Analysis of PV– and PV+ cell activity revealed that, during epochs of quiet rest (when the mouse was stationary on the treadmill), PV– cells on average displayed more frequent calcium transients, whereas there was no change in the frequency of PV+ cell transients ( Figure 8G–H ).} in-vivo-pv-minus-transient-frequency-increased⟧

Of note, this held true even if only cells that displayed at least one transient throughout the recording were included in the analysis ( Figure 8—figure supplement 1A and B ).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{Of note, this held true even if only cells that displayed at least one transient throughout the recording were included in the analysis ( Figure 8—figure supplement 1A and B ).} in-vivo-pv-minus-transient-frequency-increased — This is the same quiet-rest transient-frequency result the claim states, shown to survive restriction to cells with at least one transient.⟧

This effect is generally consistent with decreased perisomatic inhibition of excitatory cells in Kcnc1 -A421V/+ mice.

In line with this hypothesis, PV+ cells on average displayed lower amplitude transients, which could indicate that fewer APs underlie each calcium transient ( Zhang et al., 2023 ).

We also observed a decrease in the amplitude of the transients in both PV– and PV+ cells ( Figure 8I and J ).

These decreases in the frequency of PV– cell transients and the height of PV+ cell transients were not seen during epochs where the mouse was running ( Figure 8—figure supplement 1C–F ).⟦>zach claim=gap: @{These decreases in the frequency of PV– cell transients and the height of PV+ cell transients were not seen during epochs where the mouse was running ( Figure 8—figure supplement 1C–F ).} No claim records the state-dependence of the in vivo effects — that they are absent during running epochs.⟧

We also observed no differences in the overall percentage of cells that were active in Kcnc1 -A421V/+ relative to WT mice ( Figure 8—figure supplement 1G and H ).⟦>zach claim=gap: @{We also observed no differences in the overall percentage of cells that were active in Kcnc1 -A421V/+ relative to WT mice ( Figure 8—figure supplement 1G and H ).} No claim records that the overall percentage of active cells is unchanged between genotypes.⟧

Spontaneous seizures and premature lethality in Kcnc1 -A421V/+ mice To further investigate the nature of the events observed in Kcnc1- A421V/+ mice during in vivo 2P calcium imaging, we performed continuous video electroencephalogram (EEG) monitoring for periods of 2–7 days in young adult (P24–48) WT and Kcnc1 -A421V/+ mice ( Figure 9 ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{Spontaneous seizures and premature lethality in Kcnc1 -A421V/+ mice To further investigate the nature of the events observed in Kcnc1- A421V/+ mice during in vivo 2P calcium imaging, we performed continuous video electroencephalogram (EEG) monitoring for periods of 2–7 days in young adult (P24–48) WT and Kcnc1 -A421V/+ mice ( Figure 9 ).} spontaneous-seizures-and-sudep-kcnc1⟧

In eight of 12 Kcnc1 -A421V/+ mice, we observed clear convulsive seizures, including tonic, clonic, and tonic-clonic limb movements with loss of consciousness and fall, associated with an electrographic correlate (in the mice that exhibited seizures, the average seizure frequency was 0.62±0.24 per day and event duration was 32.4±15.7 s; Figure 9A and B ; Video 2 ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{In eight of 12 Kcnc1 -A421V/+ mice, we observed clear convulsive seizures, including tonic, clonic, and tonic-clonic limb movements with loss of consciousness and fall, associated with an electrographic correlate (in the mice that exhibited seizures, the average seizure frequency was 0.62±0.24 per day and event duration was 32.4±15.7 s; Figure 9A and B ; Video 2 ).} spontaneous-seizures-and-sudep-kcnc1⟧

We did not detect any seizures or other EEG abnormalities in four of four recorded WT control mice ( Figure 9C ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{We did not detect any seizures or other EEG abnormalities in four of four recorded WT control mice ( Figure 9C ).} spontaneous-seizures-and-sudep-kcnc1⟧

As in our 2P in vivo calcium imaging experiments, we also observed brief diffuse jerks involving the face and limbs associated with large-amplitude spikes on the EEG Kcnc1 -A421V/+ mice in recorded ( Figure 9A–C ; Video 2 ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{As in our 2P in vivo calcium imaging experiments, we also observed brief diffuse jerks involving the face and limbs associated with large-amplitude spikes on the EEG Kcnc1 -A421V/+ mice in recorded ( Figure 9A–C ; Video 2 ).} spontaneous-seizures-and-sudep-kcnc1⟧

In 8 of 12 recorded Kcnc1 -A421V/+ mice, we observed brief runs of epileptiform spikes without apparent behavioral correlate (seizure duration was 14.5±0.7 s; Figure 9C ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{In 8 of 12 recorded Kcnc1 -A421V/+ mice, we observed brief runs of epileptiform spikes without apparent behavioral correlate (seizure duration was 14.5±0.7 s; Figure 9C ).} spontaneous-seizures-and-sudep-kcnc1⟧

Interestingly, we were also able to capture four Kcnc1 -A421V/+ seizure-induced sudden death events on video-EEG, with two additional occurrences with video only.

In each case, sudden death was directly preceded by a generalized tonic-clonic seizure with hindlimb extension, whereas nonfatal seizures did not lead to the hindlimb extension associated with the tonic phase of generalized seizures ( Figure 9A and B ).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{In each case, sudden death was directly preceded by a generalized tonic-clonic seizure with hindlimb extension, whereas nonfatal seizures did not lead to the hindlimb extension associated with the tonic phase of generalized seizures ( Figure 9A and B ).} spontaneous-seizures-and-sudep-kcnc1⟧

Overall, our in vivo studies reveal that the Kcnc1 -A421V/+ mouse recapitulates core features of KCNC1 DEE with a range of seizure types, including myoclonic seizures.

Figure 9. Kcnc1 -A421V/+ mice exhibit spontaneous seizures and seizure-induced death.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{Figure 9. Kcnc1 -A421V/+ mice exhibit spontaneous seizures and seizure-induced death.} spontaneous-seizures-and-sudep-kcnc1⟧

( A ) Representative example trace of the electroencephalogram (EEG) collected from an adult Kcnc1 -A421V/+ mouse during a nonfatal seizure.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{( A ) Representative example trace of the electroencephalogram (EEG) collected from an adult Kcnc1 -A421V/+ mouse during a nonfatal seizure.} spontaneous-seizures-and-sudep-kcnc1⟧

After the seizure-related spike-wave discharges, there are large spikes that are associated with diffuse whole-body jerks.

( B ) Representative generalized tonic-clonic seizure resulting in seizure-induced sudden death in a Kcnc1 -A421V/+ mouse.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{( B ) Representative generalized tonic-clonic seizure resulting in seizure-induced sudden death in a Kcnc1 -A421V/+ mouse.} spontaneous-seizures-and-sudep-kcnc1⟧

( C ) Raster plot indicating nonfatal seizures (blue bars), seizure-induced sudden death (red bars), interictal runs of spikes (green bars) without clear behavior manifestation, and periods of repetitive myoclonic seizures (yellow shading) for each mouse examined (N=12).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{( C ) Raster plot indicating nonfatal seizures (blue bars), seizure-induced sudden death (red bars), interictal runs of spikes (green bars) without clear behavior manifestation, and periods of repetitive myoclonic seizures (yellow shading) for each mouse examined (N=12).} spontaneous-seizures-and-sudep-kcnc1⟧

Recordings in wild-type (WT) (N = 4) control mice did not show epileptic seizures or runs of spikes.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{Recordings in wild-type (WT) (N = 4) control mice did not show epileptic seizures or runs of spikes.} spontaneous-seizures-and-sudep-kcnc1 — The claim states that all four WT controls showed zero seizures on video-EEG.⟧

Video 2. Example seizure with myoclonic jerks.


## discussion

Discussion The recurrent pathogenic variant KCNC1 -p.A421V leads to DEE characterized by treatment-resistant epilepsy with onset in the first year of life with multiple seizure types, including myoclonic seizures, moderate to severe global developmental delay/intellectual disability, and variably present but mild nonprogressive ataxia.

Further elucidation of the mechanistic links between KCNC1 variants, Kv3.1 subunit-containing K + channel dysfunction, impairments in the intrinsic excitability of Kv3.1-expressing neurons, and synaptic and circuit neurophysiology is critical toward clarification of underlying disease pathomechanisms and development of potential therapeutic intervention.

Our study reports the generation of a mouse model of KCNC1 DEE and determines the physiological mechanisms of disease at the level of ion channels, single neuron intrinsic excitability, and synaptic neurotransmission, as well as in circuits in vivo , within epilepsy-related brain regions.

Kv3.1 expression and function Kv3.1 is specifically expressed in high-frequency firing neurons throughout the nervous system, including PV-INs in the neocortex, hippocampus, amygdala, and basal ganglia, as well as cells of the reticular thalamic nucleus, and Purkinje cells, granule cells, and molecular layer interneurons of the cerebellum ( Chow et al., 1999 ).

Due to rapid activation and deactivation kinetics and unique voltage dependence (more positively shifted than any other K + channel), Kv3.1 and other members of the Kv3 family (Kv3.2, Kv3.3) are associated with neurons that generate APs at particularly high frequencies > 200 Hz ( Erisir et al., 1999 ; Kaczmarek and Zhang, 2017 ).

Kv3.1 knockout (Kv3.1 –/– ) animals have previously been used to investigate the functional contribution of Kv3.1 to neuronal spiking.

These mice have reduced body weight and altered sensory/motor function, as we observed in Kcnc1 -A421V/+ mice, but do not display spontaneous seizures ( Ho et al., 1997 ).

The identified impairments in intrinsic neuronal excitability of Kv3.1-expressing neurons were relatively subtle in Kv3.1 knockout mice: RTN neurons from Kv3.1 –/– mice exhibited slightly wider APs and a use-dependent spike broadening that produced a mild impairment in AP frequency, but, overall, there seemed to be functional and/or genetic compensatory upregulation of other Kv3 subfamily members in response to Kv3.1 deletion ( Porcello et al., 2002 ).

Mice lacking Kv3.2 (Kv3.2 –/– ), the other Kv3 family member highly expressed in PV-INs and which has near-identical biophysical properties, perhaps exhibit a somewhat more similar phenotype to that identified in the Kcnc1 -A421V/+ mice, with impaired excitability of neocortical PV-INs and spontaneous seizures observed in a subset of mice ( Lau et al., 2000 ).

Overall, for mechanistic reasons that are not yet completely clear, it seems that the heterozygous Kcnc1 -A421V/+ mice reported here have a more severe phenotype than either Kv3.1 or Kv3.2 null mice.

One possibility is that compensation shown to occur in knockout mice might not occur with heterozygous expression of a missense variant (i.e. the variant ‘escapes’ compensation).

Our data further supports the conclusion that the Kv3.1-A421V variant exerts a dominant-negative action on trafficking, as well as a possible additional effect on gating of Kv3.1/Kv3.2 heteromultimeric channels that do successfully traffic to the membrane.

In contrast, Kv3.1 and Kv3.2 knockout mice influence only one Kv3 subfamily member (and drive compensatory upregulation of each other).

Our results show that the A421V variant leads to decreased Kv3-like current in nucleated macropatches from neocortical PV-INs, as well as impaired cell surface expression of Kv3.1 in neocortical PV-INs.

Future studies should further clarify the precise mechanism whereby this missense variant impacts trafficking of and incorporation into heteromultimeric Kv3 channels in various subcellular compartments of PV-INs and other Kv3.1-expressing cells.

We found that Kv3.1-expressing neocortical PV-INs and cells of the RTN, but not excitatory cells (which do not express Kv3.1), were hypoexcitable in Kcnc1- A421V/+ relative to WT mice, generating fewer APs in response to depolarizing current injections.

At P16-21, PV-INs exhibited a small but significant depolarization in resting membrane potential, which may reflect delayed development of PV-INs in Kcnc1 -A421V/+ mice ( Goldberg et al., 2011 ), be a direct result of altered potassium channel function, and/or could represent a compensatory response to intrinsic hypoexcitability.

Beyond a role for PV-positive Kv3.1-expressing fast-spiking neurons, the extent to which specific cellular populations (e.g. cerebral cortex interneurons vs. neurons of the RTN) contributes to the overall epileptic/behavioral phenotype of the Kcnc1 -A421V/+ mice remains unclear.

Future studies using focal Cre injection or region-specific Cre drivers to express the A421V variant in a cell-type and/or region-specific restricted manner could be helpful for addressing these remaining questions.

Hypofunction of PV-INs has been associated with various types of epilepsy, including, most notably, Dravet syndrome, a DEE driven by loss-of-function variants in SCN1A encoding the voltage-gated sodium channel subunit Na V 1.1. Hence, Dravet syndrome and KCNC1 DEE converge on specific impairment of cerebral cortex GABAergic inhibitory interneurons, and on PV-INs in particular.

Yet, there are likely important differences between these syndromes which may explain the divergent clinical presentation in patients ( Clatot et al., 2024 ).

For one, deficits in Kv3.1 in KCNC1 DEE may be differentially compensated by other Kv3 isoforms (perhaps remaining uncompensated) when compared to a possible compensation for reduced Na V 1.1 in Dravet syndrome by other voltage-gated sodium channel α subunits.

Second, there is a differential cell type-specific expression pattern between Kv3.1 and Na V 1.1 in the cerebral cortex, with Na V 1.1 being also expressed in non-fast-spiking interneurons such as somatostatin and VIP-expressing interneurons ( Tai et al., 2014 ; Rubinstein et al., 2015 ; Goff and Goldberg, 2019 ), whereas Kv3.1 is largely specific for PV-INs.

Yet, Kv3.1 is more prominently expressed in superficial layers of mouse neocortex, with Kv3.2 more prominently expressed in deep layer PV-INs, while Na V 1.1 appears to be expressed in PV-INs across layers of the neocortex.

Our results here also indicate another point of divergence between mechanisms of Dravet syndrome and KCNC1 DEE: in adult Kcnc1 -A421V/+ mice, we observed an increase in the magnitude and altered paired-pulse ratio of PV-IN-mediated inhibitory postsynaptic currents relative to WT mice accompanied by no change in failure rate, which contrasts with the increased rate of failure and prolonged synaptic latency that we previously observed in PV-IN-mediated neurotransmission in Dravet syndrome ( Scn1a +/– ) mice ( Kaneko et al., 2022 ).

We interpret the augmentation in postsynaptic current magnitude alongside reduced paired-pulse ratio observed in young adult (P32–42, after epilepsy onset) Kcnc1 -A421V/+ mice to be generally consistent with a role for Kv3.1 in regulating neurotransmitter release by controlling spike-evoked calcium via presynaptic AP width, as shown previously ( Goldberg et al., 2005 ), although there could also be roles for secondary dysregulation of or compensatory alterations in other determinants of synaptic transmission (such as GABA receptor expression).

We did not find alterations in inhibitory synaptic neurotransmission at the P16–21 time point, despite the fact that PV-INs already exhibited markedly impaired intrinsic excitability and reduced magnitude of somatic voltage-gated K + currents.

These results may indicate that the physiological contribution of Kv3.1 in different subcellular regions (i.e. soma, dendrite, axon, synaptic terminal, etc.) and its corresponding role in regulating the associated physiological phenomena (AP generation, propagation, and neurotransmitter release) evolves over development.

For example, the demonstrated impairment in trafficking of Kv3.1-A421V variant subunit containing Kv3 channels implies that distal synaptically localized Kv3 channels may not contain variant subunits at early time points and hence local AP waveform at the synapse might remain largely normal via residual Kv3 channels containing WT Kv3.1 and/or Kv3.2 (or Kv3.3) subunits.

A more detailed mechanistic explanation for this age-dependent effect would provide further insight into disease pathomechanisms and could explain why the epilepsy phenotype appears at/around the time of weaning and increases in severity in this mouse model (in stark contrast to Scn1a +/– mice, where epilepsy severity decreases with age).

However, this would require a detailed exploration of the specific composition of heterotetrameric Kv3 channels in WT vs. Kcnc1 -A421V/+ mice in various subcellular compartments and across development, as suggested above.

In this study, we focused on PV-INs and excitatory neurons in somatosensory neocortical layer II-IV, PV-INs of layer V, and PV-positive neurons of the reticular thalamic nucleus – epilepsy-linked brain regions – due to the prominent epilepsy phenotype and seizure-related early mortality observed in Kcnc1- A421V/+ mice.

However, there are many other cellular populations across various brain regions that express Kv3.1, which could also be examined in future studies.

As noted above, given that our mouse model expresses the knock-in A421V variant under the control of Cre recombinase, we are well positioned to explore how cell type and developmental timing of altered Kv3.1 function might contribute to overall behavior phenotype.

Kcnc1 -A421V/+ mice recapitulate the core phenotype of KCNC1 epilepsy seen in human patients Patients harboring the KCNC1 -p.A421V variant exhibit treatment-resistant epilepsy with various seizure types, including myoclonic, focal, atypical absence, and generalized tonic-clonic seizures with onset in the first year of life ( Cameron et al., 2019 ; Park et al., 2019 ).

The novel Kcnc1 -A421V/+ mouse model well captured the range of seizure phenotypes seen in human patients: spontaneous seizures with different behavioral manifestations were observed, including myoclonic seizures, focal convulsive seizures, and generalized tonic-clonic seizures (those associated with hindlimb extension leading to sudden death).

We directly observed abnormal neocortical neural activity in the Kcnc1 -A421V/+ mice in our in vivo 2P calcium imaging experiments accompanied by behavioral correlates of myoclonic seizures, demonstrating this mouse represents a potentially useful model for study of mechanisms of spontaneous seizures – including myoclonic seizures – using 2P calcium imaging in awake, behaving mice.

These experiments revealed that apparent myoclonic seizures were associated with hypersynchronous paroxysmal discharges seen across all neurons within the field of view, with prominent activation of the neuropil.

Although we separately labeled fast-spiking PV-INs and other cells in our in vivo imaging experiments, we did not observe cell type-specific differences in recruitment of PV-INs and non-PV cells, which might indicate a causal relationship between aberrant PV-IN activity and the hypersynchronous discharge.

However, it is perhaps more likely that such seizures engaged diffuse brain networks, and the observed hypersynchronous discharges (and neuropil signal) were driven by abnormal distal activity.

Nevertheless, these otherwise brief and intermittent events were clearly identified via 2P imaging which led to subsequent EEG studies confirming such events to be seizures.

All Kcnc1 -A421V/+ mice exhibited hypersynchronous discharges in our initial 2P experiments; yet, we did not observe such hypersynchronous discharges in all mice in which GCaMP expression was restricted to the soma.

The basis of this apparent discrepancy remains unclear but may support the conclusion that such events are generated distally and recruit the neurites of cells in the imaging field.

Future studies should expand on the in vivo imaging and EEG completed here to more thoroughly investigate the cellular and network architecture of the neural activity underlying the spontaneously occurring myoclonic seizures.

Kcnc1 -A421V/+ mice may prove to be a particularly tractable model for the study of myoclonic seizures.

Beyond seizures, human patients harboring KCNC1 variants show moderate to severe developmental delay and intellectual disability ( Cameron et al., 2019 ; Park et al., 2019 ).

Young Kcnc1 -A421V/+ mice showed developmental differences in body/brain weights, and although we did not detect other gross impairments in developmental milestones between P5 and P15, which aligns with the expected developmental expression pattern of Kv3.1 and onset of fast-spiking around P15 ( Okaty et al., 2009 ; Goldberg et al., 2011 ), adult (>P35) Kcnc1 -A421V/+ mice exhibited cognitive impairment in both the Y-maze and Barnes maze test.

These early developmental tests may have limited sensitivity to detect early subtle differences, and future studies should expand on this work with additional testing of cognitive, motor, social, and other behaviors.

The A421V Kcnc1 variant leads to a loss of voltage-gated potassium channel function in PV-INs Previous studies have reported that A421V is a loss-of-function variant when examined in cell systems ( Cameron et al., 2019 ; Park et al., 2019 ).

However, such work is conflicting as to the exact mechanism, with one paper showing evidence for a dominant-negative effect and another paper finding no evidence for dominant-negative action of the A421V variant.

Differences in results obtained in Xenopus oocytes vs. mammalian cells may relate to culture conditions such as temperature, which is known to affect protein folding and trafficking.

Our outside-out nucleated macropatch recordings of somatic voltage-gated K + currents in brain slice showed clear ∼50% reduction in K + current density in PV-INs (but not excitatory cells) without changes in the biophysical properties of gating.

In our examination of surface Kv3.1, we found a reduction in the amount of Kv3.1 that reaches the membrane in PV-INs from Kcnc1 -A421V/+ mice.

While we cannot rule out the possibility that some Kv3 tetramers at the cell surface contain Kv3.1-A421V subunits and act to decrease channel conductance (as suggested by our HEK cell recordings), our data is consistent with the view that the variant acts at least in part via incorporation of Kv3.1-A421V subunits into heterotetrameric Kv3 channels (likely in the endoplasmic reticulum) with impaired trafficking to the membrane.

Consistent with this, a previous study identified A421V as exerting only slight steric hindrance relative to other developmental encephalopathy-causing KCNC1 variants, supporting the conclusion that the profound impact of this variant on recorded currents is likely due mainly to impaired trafficking, potentially with some contribution via an impact on gating ( Li et al., 2021 ).

Similar structural approaches may also help better determine the mechanism of trafficking deficiency and the extent to which A421V channels impair the trafficking of heteromultimeric Kv3 channels containing WT Kv3.1 and/or Kv3.2 subunits.

Kv3.1 as a drug target in epilepsy Given the powerful influence of Kv3 channels on the excitability of neocortical PV-INs and neurons of the cerebellum, pharmacological modulators of Kv3.1 have been proposed as potential treatment for a range of neurological and psychiatric conditions, including in a mechanistically targeted fashion for patients with KCNC1 -related disorders such as EPM7 ( Rosato-Siri et al., 2015 ; Brown et al., 2016 ; Boddum et al., 2017 ; Chambers et al., 2017 ; Munch et al., 2018 ; Feng et al., 2024 ).

Previous reports showed that the Kv3 positive modulator AUT-1 and related compounds facilitate greater firing frequency and spiking reliability of fast-spiking cells ( Rosato-Siri et al., 2015 ; Brown et al., 2016 ; Boddum et al., 2017 ; Chambers et al., 2017 ; Munch et al., 2018 ; Feng et al., 2024 ).

Our study did not investigate the impact of Kv3.1 modulators in Kcnc1 -A421V/+ mice.

Yet, considering the decreased cell surface expression of Kv3.1 in PV-INs from Kcnc1 -A421V/+ mice, one might predict limited efficacy of a small-molecule channel activator, unless such compounds could exert therapeutic effect via action on WT Kv3 channels not containing variant Kv3.1-A421V subunits.

On the other hand, genetic approaches to either knock down expression of the A421V variant (perhaps using an antisense oligonucleotide) or boost expression levels of the WT Kv3.1 could be explored.

Limitations of the study We provide evidence for a strong loss of total potassium current density and deficits in excitability in Kcnc1 -A421V/+ PV-INs relative to WT, with the most severe alterations to excitability observed for PV-INs in superficial neocortical layers likely driven by a high relative expression of Kv3.1 vs. Kv3.2 in these cells ( Chow et al., 1999 ).

While we also provided immunohistochemical evidence that variant Kv3.1 leads to impaired membrane trafficking of Kv3.1, the molecular details underlying how the variant induces an overall loss of potassium channel function remain to be definitively determined.

For example, it is unknown what relative proportion of A421V-containing heterotetramers reach the cell surface, and, for any channels that do, it is yet unclear the extent to which such channels functionally gate and flux potassium.

Considering that the Kcnc1 -A421V/+ mouse is significantly more severely affected in cellular and behavioral phenotype than Kv3.1 and 3.2 knockout mice, and that layer V PV-INs exhibit less severe impairment than layer II-IV PV-INs, we suspect that Kv3.1 A421V variant subunits exert a dominant-negative influence on Kv3 channel cell surface expression and function, i.e., such variants impact all PV-IN Kv3.1/Kv3.2 heteromultimeric channels containing one or more Kv3.1 A421V variant subunits.

This could be compounded by potential electrophysiological dysfunction of Kv3 channels containing Kv3.1 A421V variants that do traffic to the membrane.

We focused our study on the global impact of the Kcnc1 -A421V variant on mouse development, epilepsy, and neuronal physiology of selected neuron types in epilepsy-linked brain regions, using Actb-Cre to drive global expression from the blastocyst stage so as to best model the human condition.

Future work using cell type-specific Cre drivers or Cre delivery to restricted cell types and/or brain regions will enable greater mechanistic clarity in linking cell type and brain region to specific aspects of the mouse phenotype, including epilepsy and non-epilepsy comorbidities of cognitive and motor impairment.

However, given the early onset of neurological dysfunction in our mice, specific expression of the variant using, for example, PV-Cre mice might not yield greater mechanistic insight, as PV itself is not expressed at appreciable levels until at/beyond P10 in mice and hence efficient recombination and expression of the Kcnc1 -p.A421V variant in PV-INs will likely not faithfully reproduce the appropriate developmental expression pattern.

Conclusion In summary, we report a mouse model that recapitulates the core features of KCNC1 DEE due to the recurrent K + channel variant KCNC1 -p.A421V.

Kcnc1 -A421V/+ mice exhibit epilepsy with multiple seizure types, including myoclonic seizures, as well as cognitive impairment.

This was associated with a pattern of specific impairments in intrinsic excitability and synaptic transmission consistent with Kv3 dysfunction, observed in Kv3.1-expressing neurons linked to epilepsy, including neocortical PV-INs and neurons of the reticular thalamic nucleus, but not excitatory cells.

Future studies and ongoing therapeutic development promise to expand this mechanistic understanding in pursuit of improved outcomes for patients with this severe yet currently incurable and untreatable disorder.


## captions

=== Figure 1 === Figure 1. Design of a novel mouse model of KCNC1 developmental and epileptic encephalopathy.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{=== Figure 1 === Figure 1. Design of a novel mouse model of KCNC1 developmental and epileptic encephalopathy.} a421v-mice-die-before-122d⟧

( A ) Design and structure of the conditional Kcnc1 -A421V allele.

Upon Cre-mediated recombination, the inserted wild-type (WT) coding sequence (CDS) flanked by LoxP sites is removed and the A421V variant inserted into exon 2 is expressed.

( B ) Sequencing results indicate successful targeting of c.1262C>T to introduce the heterozygous A421V variant.

( C ) PCR confirmation of two HET founders ( Kcnc1 -A421V/+) and two WT littermates. 167 bp, WT allele fragment; 207 bp, floxed allele fragment.

( D ) Breeding strategy to generate control and experimental mice in which the Kcnc1 -A421V variant is expressed globally and PV cells are fluorescently labeled for targeted recording.

( E ) Survival plot of WT (N=46; black ) and Kcnc1 -A421V/+ (N=33; green ) mice. ***p<0.001 by log-rank Mantel-Cox curve comparison.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{( E ) Survival plot of WT (N=46; black ) and Kcnc1 -A421V/+ (N=33; green ) mice. ***p<0.001 by log-rank Mantel-Cox curve comparison.} a421v-mice-die-before-122d⟧

[panels detected: a, b, c, d, e] === Figure 1s1 === Figure 1—figure supplement 1. Pvalb-tdTomato reporter effectively labels parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) in wild-type (WT) and Kcnc1 -A421V/+ mice.⟦>zach claim=gap: @{[panels detected: a, b, c, d, e] === Figure 1s1 === Figure 1—figure supplement 1. Pvalb-tdTomato reporter effectively labels parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) in wild-type (WT) and Kcnc1 -A421V/+ mice.} No claim records the validation of the Pvalb-tdTomato reporter as a faithful label for PV-INs in either genotype.⟧

( A ) Representative immunohistochemistry images for parvalbumin in WT (top row) and Kcnc1 -A421V/+ (bottom row) mice (postnatal day [P]21–33) showing a high degree of overlap between the tdTomato reporter and parvalbumin expression.

The asterisk indicates rare cells that are tdTomato + , but parvalbumin – .

The arrowhead indicates cells that are parvalbumin + but tdTomato – .

Scale bar, 100 μm.

( B ) Counts of PV cells per unit area (mm 2 ) are not different between WT and Kcnc1 -A421V/+ mice (N=3 mice/genotype).⟦>zach claim=gap: @{( B ) Counts of PV cells per unit area (mm 2 ) are not different between WT and Kcnc1 -A421V/+ mice (N=3 mice/genotype).} No claim records that neocortical PV cell density is unchanged in Kcnc1-A421V/+ mice.⟧

( C ) Sensitivity rate (probability of a cell being tdTomato + if it is parvalbumin + ; N = 6 mice/genotype).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( C ) Sensitivity rate (probability of a cell being tdTomato + if it is parvalbumin + ; N = 6 mice/genotype).} a421v-kv31-membrane-trafficking-impaired⟧

( D ) False-positive probability (proportion of all tdTomato + cells that are parvalbumin negative; N = 6 mice/genotype).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) False-positive probability (proportion of all tdTomato + cells that are parvalbumin negative; N = 6 mice/genotype).} a421v-kv31-membrane-trafficking-impaired⟧

[panels detected: a, b, c, d] === Figure 1s2 === Figure 1—figure supplement 2. Early postnatal development of Kcnc1 -A421V/+ mice.⟦>zach claim=b7a7c44d-b512-45b8-a790-a1b2c3b216e7: @{[panels detected: a, b, c, d] === Figure 1s2 === Figure 1—figure supplement 2. Early postnatal development of Kcnc1 -A421V/+ mice.} a421v-weight-reduced-milestones-normal⟧

( A ) Representative example image showing littermate wild-type (WT) and Kcnc1 -A421V/+ mice at postnatal day 21. ( B ) Average body weights for WT (N=10; black ) and Kcnc1 -A421V/+ (N=11; green ) at postnatal days 7, 14, and 21. ( C ) Average brain weights for WT and Kcnc1 -A421V/+ mice at postnatal days 7 (WT, N=2; Kcnc1 -A421V/+, N=6), 14 (WT, N=3; Kcnc1 -A421V/+, N=3), and 21 (WT, N=4; Kcnc1 -A421V/+, N=6).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( A ) Representative example image showing littermate wild-type (WT) and Kcnc1 -A421V/+ mice at postnatal day 21. ( B ) Average body weights for WT (N=10; black ) and Kcnc1 -A421V/+ (N=11; green ) at postnatal days 7, 14, and 21. ( C ) Average brain weights for WT and Kcnc1 -A421V/+ mice at postnatal days 7 (WT, N=2; Kcnc1 -A421V/+, N=6), 14 (WT, N=3; Kcnc1 -A421V/+, N=3), and 21 (WT, N=4; Kcnc1 -A421V/+, N=6).} a421v-kv31-membrane-trafficking-impaired⟧

Note that for both B and C, error bars depicting SEM are present, but do not appear beyond the symbols.

( D ).

Onset of developmental and motor benchmarks for WT (N=7) and Kcnc1 -A421V/+ (N=10) mice.⟦>zach claim=b7a7c44d-b512-45b8-a790-a1b2c3b216e7: @{Onset of developmental and motor benchmarks for WT (N=7) and Kcnc1 -A421V/+ (N=10) mice.} a421v-weight-reduced-milestones-normal — This panel displays the developmental and motor benchmark onsets the claim reports as unaltered.⟧

( E ) Onset of startle reflex and other typical behavioral/motor milestones in WT (N=7) and Kcnc1 -A421V/+ (N=10) mice. ***p<0.001 by mixed-effects analysis followed by Sidak’s multiple comparisons post hoc test.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{( E ) Onset of startle reflex and other typical behavioral/motor milestones in WT (N=7) and Kcnc1 -A421V/+ (N=10) mice. ***p<0.001 by mixed-effects analysis followed by Sidak’s multiple comparisons post hoc test.} a421v-mice-die-before-122d⟧

[panels detected: a, b, c, d, e] === Figure 2 === Figure 2. Impaired cognitive function in Kcnc1 -A421V/+ mice.⟦>zach claim=a2af2706-be34-44a7-b65b-6a11ae98c2a8: @{[panels detected: a, b, c, d, e] === Figure 2 === Figure 2. Impaired cognitive function in Kcnc1 -A421V/+ mice.} Inhibitory dysfunction in Kcnc1-A421V/+ mice emerges progressively by young adulthood.⟧

( A ) Example pathways for wild-type (WT) (black) and Kcnc1 -A421V/+ (green) during the 4-day acquisition phase of the Barnes maze test.

Blue dot indicates the escape hole.

( B ) Average escape latency during the acquisition phase for WT (black; N=15) and Kcnc1 -A421V/+ (green; N=13) mice.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{( B ) Average escape latency during the acquisition phase for WT (black; N=15) and Kcnc1 -A421V/+ (green; N=13) mice.} a421v-spatial-learning-working-memory-impaired — This panel displays the Barnes maze acquisition escape latencies the claim reports as significantly longer in mutants.⟧

( C ) Group data for time spent in target quadrant during probe trial of Barnes maze.

(D–F) Group data for WT (black; N=16) and Kcnc1 -A421V/+ mice (green; N=15) during the Y-maze test for spatial working memory.⟦>zach claim=ae5f131c-edc3-4345-96c1-2f43609dbb3e: @{(D–F) Group data for WT (black; N=16) and Kcnc1 -A421V/+ mice (green; N=15) during the Y-maze test for spatial working memory.} a421v-spatial-learning-working-memory-impaired — These panels display the Y-maze spatial working memory data the claim reports.⟧

(D) Spontaneous alternation percentage.

( E ) Total number of arm entries.

( G ) Total distance traveled.

Data are shown as mean ± SEM and were analyzed by two-way repeated-measures ANOVA with Tukey’s post hoc test ( B ) and unpaired t-test ( D–F ).

Significance is denoted as *p<0.05 or **p<0.01. [panels detected: a, b, c, d, e, f, g] === Figure 3 === Figure 3. Parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from Kcnc1 -A421V/+ mice exhibit attenuated voltage-gated potassium channel currents and impaired membrane Kv3.1 expression.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Significance is denoted as *p<0.05 or **p<0.01. [panels detected: a, b, c, d, e, f, g] === Figure 3 === Figure 3. Parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from Kcnc1 -A421V/+ mice exhibit attenuated voltage-gated potassium channel currents and impaired membrane Kv3.1 expression.} pv-in-inhibitory-synapse-altered-adult⟧

( A ) Representative image of a cell being recorded in the outside-out nucleated macropatch configuration.

( B–C ) Example family of traces of voltage-gated K + channel currents from a PV-IN from wild-type (WT) (B, black ) and Kcnc1 -A421V/+ (C, green ) mice (postnatal day [P]16–21).

( D ) Average voltage-gated K + channel current density for WT (n=13 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=17, N=3 mice).⟦>zach claim=b29d38ec-0fb8-4a7b-863e-470dd3eb378b: @{( D ) Average voltage-gated K + channel current density for WT (n=13 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=17, N=3 mice).} pv-ins-reduced-k-current-density — This panel displays the PV-IN voltage-gated K+ current density comparison that the claim reports as significantly reduced.⟧

( E ) Maximum K + channel current density per PV-IN macropatch in WT and Kcnc1 -A421V/+ mice.

( F ) Averaged normalized plots of K + conductance relative to voltage command indicating voltage dependence of activation curves for WT and Kcnc1 -A421V/+ mice.

( G ) Average activation time constant for the voltage-gated K + channel currents relative to voltage command potential in both WT and Kcnc1 -A421V/+ mice.

( H ) Representative images of individual cortical PV-INs from WT and Kcnc1 -A421V/+ mice stained for Kv3.1 (green).

Plasma membrane (PM), nucleus (nuc), and cytosol (cyt) are indicated in the top left panel.

Note the markedly less pronounced Kv3.1 intensity in the plasma membrane in each of the Kcnc1 -A421V/+ examples and more prominent cytosolic labeling (presumably corresponding to endoplasmic reticulum).

( I ) Group quantification of ratio of membrane to cytosolic Kv3.1 for WT (n=49 cells, N=6 mice) and Kcnc1 -A421V/+ (n=48 cells, N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( I ) Group quantification of ratio of membrane to cytosolic Kv3.1 for WT (n=49 cells, N=6 mice) and Kcnc1 -A421V/+ (n=48 cells, N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

Mice were between the ages of P24 and P33. Data are shown as mean ± SEM or individual data points, and significance was determined using either repeated-measures two-way ANOVA or unpaired t-test where ***p<0.001. [panels detected: a, b, c, d, e, f, g, h, i] === Figure 3s1 === Figure 3—figure supplement 1. Loss of potassium current density in A421V-expressing HEK cells.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Mice were between the ages of P24 and P33. Data are shown as mean ± SEM or individual data points, and significance was determined using either repeated-measures two-way ANOVA or unpaired t-test where ***p<0.001. [panels detected: a, b, c, d, e, f, g, h, i] === Figure 3s1 === Figure 3—figure supplement 1. Loss of potassium current density in A421V-expressing HEK cells.} a421v-mice-die-before-122d⟧

( A–C ) Example Kv3.1 currents in HEK cells expressing wild-type (WT) (A; black), a 50:50 mixture of WT and A421V (B; blue), and A421V ( C ; green) Kv3.1 subunits.

Voltage command protocol shown below WT example.

( D ) Average current density for WT (black), WT+A421V (blue), and A421V (green).

Note that the A421V variant leads to a profound loss of function.

( E ) Average normalized conductance relative to membrane potential for WT (black), WT+A421V (blue), and A421V (green).

G-V curves for the A421V variant should be interpreted with caution as the recorded currents are exceedingly small and cannot be easily differentiated from the small endogenous delayed rectifier potassium currents known to be present in HEK cells.

[panels detected: a, b, c, d, e] === Figure 4 === Figure 4. Impaired parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) intrinsic excitability in juvenile and adult Kcnc1-A421V/+ mice.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{[panels detected: a, b, c, d, e] === Figure 4 === Figure 4. Impaired parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) intrinsic excitability in juvenile and adult Kcnc1-A421V/+ mice.} pv-in-ap-waveform-altered-downstroke-apd50⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical PV-IN recorded in the whole-cell configuration to characterize intrinsic excitability.

( B–C ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA.

The inset shows an expanded view of APs generated in response to the 400 pA current injection in both genotypes.

( D ) Average relationship between PV-IN AP frequency in response to a range of current injections for juvenile WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=36 cells, N = 12 mice).⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{( D ) Average relationship between PV-IN AP frequency in response to a range of current injections for juvenile WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=36 cells, N = 12 mice).} pv-ins-impaired-maximal-firing — This panel displays the juvenile PV-IN frequency-current relationship the claim reports as impaired.⟧

( E ) Representative overlaid examples of single APs and the corresponding phase plots for juvenile WT ( black ) and Kcnc1 -A421V/+ ( green ) PV-INs.

(F) Representative images for a layer IV neocortical PV-IN from an adult mouse.

( G–H ) Representative example traces displaying intrinsic excitability in adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.

Inset shows an expanded view of APs induced by the 400 pA current step.

( I ) Average relationship between PV-IN AP frequency and current injection for adult WT (n = 14 cells, N = 3 mice) and Kcnc1 -A421V/+ mice (n=17 cells, N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( I ) Average relationship between PV-IN AP frequency and current injection for adult WT (n = 14 cells, N = 3 mice) and Kcnc1 -A421V/+ mice (n=17 cells, N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

( J ).

Representative overlaid examples of single APs and the corresponding phase plots for adult WT ( black ) and Kcnc1 -A421V/+ ( green ) PV-INs.

Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.} a421v-mice-die-before-122d⟧

[panels detected: a, b, c, d, e, f, g, h, i, j] === Figure 4s1 === Figure 4—figure supplement 1. Intrinsic physiology of neocortical layer II-IV parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) remains impaired when resting membrane potential is not normalized.⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{[panels detected: a, b, c, d, e, f, g, h, i, j] === Figure 4s1 === Figure 4—figure supplement 1. Intrinsic physiology of neocortical layer II-IV parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) remains impaired when resting membrane potential is not normalized.} pv-ins-impaired-maximal-firing — The claim states that PV-IN maximal firing is impaired, which is the effect this supplement reports as surviving without membrane-potential normalization.⟧

( A–B ) Representative example traces showing spiking in layer V neocortical PV-INs from wild-type (WT) (A, black) and Kcnc1 -A421V/+ ( B , green) mice in response to depolarizing current injections.

( C ) Average PV-IN spiking frequency relative to current injection.

A significant interaction effect between genotype and current injection was observed (***p<0.001) by two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{A significant interaction effect between genotype and current injection was observed (***p<0.001) by two-way ANOVA.} a421v-mice-die-before-122d⟧

Data are shown as mean ± SEM, and significance of post hoc comparisons (*p<0.05) by repeated-measures two-way ANOVA followed by Sidak’s multiple comparisons test.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM, and significance of post hoc comparisons (*p<0.05) by repeated-measures two-way ANOVA followed by Sidak’s multiple comparisons test.} pv-in-inhibitory-synapse-altered-adult⟧

[panels detected: a, b, c] === Figure 4s2 === Figure 4—figure supplement 2. Subtle abnormalities in neocortical layer V parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+.⟦>zach claim=d6e81203-d9f1-4344-97d0-48ec7b6afe39: @{[panels detected: a, b, c] === Figure 4s2 === Figure 4—figure supplement 2. Subtle abnormalities in neocortical layer V parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) from juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+.} layer-v-pv-ins-subtle-impairment⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a PV-positive cell in the reticular thalamus recorded in the whole-cell configuration to characterize intrinsic excitability.

( B ) Representative example traces of action potentials (APs) generated in response to current injections of varying magnitudes.

( C ) Average hyperpolarization-induced rebound APs in postnatal day (P)16–21 wild-type (WT) (n=16 cells, N=4 mice) and Kcnc1 -A421V/+ (n=19 cells, N=5 mice) in response to various current injections from 0 to –100 pA.⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( C ) Average hyperpolarization-induced rebound APs in postnatal day (P)16–21 wild-type (WT) (n=16 cells, N=4 mice) and Kcnc1 -A421V/+ (n=19 cells, N=5 mice) in response to various current injections from 0 to –100 pA.} a421v-kv31-membrane-trafficking-impaired⟧

( D ) Frequency-current relationship shows impaired intrinsic excitability in the RT PV cells relative to depolarizing current injections (0–400 pA).

Data are shown as mean ± SEM, and significance is denoted as *p<0.05 or ***p<0.001 by repeated-measures two-way ANOVA.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM, and significance is denoted as *p<0.05 or ***p<0.001 by repeated-measures two-way ANOVA.} pv-in-inhibitory-synapse-altered-adult⟧

[panels detected: a, b, c, d] === Figure 4s3 === Figure 4—figure supplement 3. Abnormal intrinsic physiology in parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) of the reticular thalamus in Kcnc1 -A421V/+ mice.⟦>zach claim=37d23efe-7e34-48aa-82ff-b7f3ef45751c: @{[panels detected: a, b, c, d] === Figure 4s3 === Figure 4—figure supplement 3. Abnormal intrinsic physiology in parvalbumin-positive fast-spiking GABAergic inhibitory interneurons (PV-INs) of the reticular thalamus in Kcnc1 -A421V/+ mice.} rtn-neurons-impaired-excitability⟧

( A–B ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) (A, black ) and Kcnc1 -A421V/+ (B, green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA from their resting membrane potential without DC bias current (as in Figure 4 ).⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{( A–B ) Representative example traces for juvenile (postnatal day [P]16–21) wild-type (WT) (A, black ) and Kcnc1 -A421V/+ (B, green ) PV-INs generating action potentials (APs) at current injections of –100, 200, 300, and 400 pA from their resting membrane potential without DC bias current (as in Figure 4 ).} pv-in-ap-waveform-altered-downstroke-apd50⟧

( C ) Average frequency-current relationship for WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=32 cells, N = 12 mice) PV-INs with uncorrected resting membrane potential.⟦>zach claim=894a5d58-bca8-4b16-b22b-76b64db1e3e5: @{( C ) Average frequency-current relationship for WT (n=20 cells, N = 9 mice) and Kcnc1 -A421V/+ (n=32 cells, N = 12 mice) PV-INs with uncorrected resting membrane potential.} pv-ins-impaired-maximal-firing — This is the same neocortical PV-IN frequency-current impairment the claim states, measured without correcting resting membrane potential.⟧

Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.⟦>zach claim=e2edfcd1-ec01-4ec7-ba36-01ab26290d69: @{Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.} a421v-mice-die-before-122d⟧

[panels detected: a, b, c] === Figure 5 === Figure 5. Unaltered potassium currents and physiological function of excitatory neurons in postnatal day (P)16–21 Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{[panels detected: a, b, c] === Figure 5 === Figure 5. Unaltered potassium currents and physiological function of excitatory neurons in postnatal day (P)16–21 Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile⟧

( A ) Representative image of a neocortical excitatory cell being recorded in the outside-out nucleated macropatch configuration.

( B–C ) Example family of traces of voltage-gated K + channel currents from an excitatory cell from wild-type (WT) ( B , black ) and Kcnc1 -A421V/+ ( C , green ) mice.

( D ) Average voltage-gated K + channel current density for WT (n=8 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=10, N=3 mice) relative to membrane potential.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( D ) Average voltage-gated K + channel current density for WT (n=8 macropatches, N=3 mice) and Kcnc1 -A421V/+ (n=10, N=3 mice) relative to membrane potential.} excitatory-neurons-unaffected-juvenile — This panel displays the juvenile excitatory-cell K+ current density the claim reports as showing no genotype difference.⟧

(E) Peak voltage-gated K + channel current density in WT and Kcnc1 -A421V/+ mice.

( F ) Normalized voltage-dependent activation curves for WT and Kcnc1 -A421V/+ mice.

( G ) Average voltage-dependent activation time constant for the voltage-gated K + channel currents.

( H ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a layer IV neocortical excitatory cell recorded in the whole-cell configuration to characterize intrinsic excitability.

( I–J ) Representative example traces showing excitatory cell action potential (AP) generation in WT ( I , black ) and Kcnc1 -A421V/+ ( J , green ) in response to depolarizing current injections.

( K ) Average relationship between excitatory cell AP frequency in response to a range of current injections for WT (N=23 cells, N=3 mice) and Kcnc1 -A421V/+ (n=22, N=3 mice).⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{( K ) Average relationship between excitatory cell AP frequency in response to a range of current injections for WT (N=23 cells, N=3 mice) and Kcnc1 -A421V/+ (n=22, N=3 mice).} excitatory-neurons-unaffected-juvenile — This panel displays the juvenile excitatory-cell frequency-current relationship the claim reports as unchanged.⟧

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA or unpaired t-test.

[panels detected: a, b, c, d, e, f, g, h, i, j, k] === Figure 5s1 === Figure 5—figure supplement 1. Intrinsic excitability is unchanged in excitatory cells from postnatal day (P)32 to P42 Kcnc1 -A421V/+ mice.⟦>zach claim=3df55d45-b8a0-42c6-9807-0323c08e8815: @{[panels detected: a, b, c, d, e, f, g, h, i, j, k] === Figure 5s1 === Figure 5—figure supplement 1. Intrinsic excitability is unchanged in excitatory cells from postnatal day (P)32 to P42 Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-adult⟧

( A ) Representative images taken at ×10 ( left ) and ×40 ( right ) magnification of a recorded layer IV excitatory cell.

( B–C ) Representative example traces of evoked action potentials (APs) in response to depolarizing current injections in cortical layer IV excitatory neurons from wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) mice.

( D ) Average frequency-current relationship for WT (black; n=12, N=4) and Kcnc1 -A421V/+ (n=12, N=4) mice.⟦>zach claim=3df55d45-b8a0-42c6-9807-0323c08e8815: @{( D ) Average frequency-current relationship for WT (black; n=12, N=4) and Kcnc1 -A421V/+ (n=12, N=4) mice.} excitatory-neurons-unaffected-adult — This panel displays the adult (P32-42) excitatory-cell frequency-current relationship the claim reports as showing no genotype difference.⟧

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA.

[panels detected: a, b, c, d] === Figure 6 === Figure 6. Juvenile (P16–21) Kcnc1 -A421V/+ mice exhibit normal parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated inhibitory synaptic neurotransmission.⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{[panels detected: a, b, c, d] === Figure 6 === Figure 6. Juvenile (P16–21) Kcnc1 -A421V/+ mice exhibit normal parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated inhibitory synaptic neurotransmission.} pv-in-inhibitory-synapse-intact-juvenile⟧

( A ) Representative images showing simultaneous whole-cell patch-clamp recordings of cortical PV-IN and nearby excitatory cell ( left , ×10 magnification; right, ×40 magnification).

( B ) Example traces of a PV-IN and excitatory cell pair in which generation of action potentials (APs) in the PV-IN (bottom trace) is sufficient to induce clear unitary inhibitory postsynaptic potentials (uIPSPs) in the excitatory cell (arrowhead, top trace).

The inset shows an example view of the individual uIPSPs corresponding to each AP in the PV-IN.

( C–D ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both wild-type (WT) ( C ) and Kcnc1 -A421V/+ pairs of synaptically connected neurons. 10 APs were generated in the PV-IN (top trace) at 40 Hz and the uIPSCs are shown below.

The black and green traces are the averages of numerous individual sweeps shown in gray.

( E ) Probability of synaptic connection between PV-IN and excitatory cell in WT (n=21 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=5 of 43 pairs from N=11 mice).⟦>zach claim=dbc34885-27d0-4b6e-afbb-6a34befb9b98: @{( E ) Probability of synaptic connection between PV-IN and excitatory cell in WT (n=21 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=5 of 43 pairs from N=11 mice).} pv-in-inhibitory-synapse-intact-juvenile⟧

( F ) Average failure probability relative to presynaptic stimulation frequency in WT and Kcnc1 -A421V/+ neuron pairs.

( G–I ) Average uIPSC magnitude for the first five APs in WT and Kcnc1 -A421V/+ at 20 Hz ( G ), 40 Hz ( H ), and 80 Hz ( I ) stimulus frequencies.

( J–K ) Paired-pulse ratios for both WT and Kcnc1 -A421V/+ mice relative to stimulus frequency.

The ratio of second uIPSC to the first is provided in J , while K displays the average ratio of the last uIPSC to the first.

(L) Average latency from peak of presynaptic AP to peak of the uIPSC in WT and Kcnc1 -A421V/+ mice.

Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA or unpaired t-test.

[panels detected: a, b, c, d, e, f, g, h, i, j, k, l] === Figure 6s1 === Figure 6—figure supplement 1. Excitatory neuron to parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) unitary excitatory synaptic neurotransmission is unaltered in juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+ mice.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{[panels detected: a, b, c, d, e, f, g, h, i, j, k, l] === Figure 6s1 === Figure 6—figure supplement 1. Excitatory neuron to parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) unitary excitatory synaptic neurotransmission is unaltered in juvenile (postnatal day [P]16–21) Kcnc1 -A421V/+ mice.} excitatory-neurons-unaffected-juvenile — The claim states that synaptic transmission involving juvenile excitatory neurons is unaltered, which is this supplement's finding for excitatory-to-PV-IN transmission.⟧

( A ) Example traces of simultaneous recordings of a synaptically connected PV-IN and excitatory cell neuron pair in which generation of action potentials (APs) in the excitatory cell leads to unitary excitatory postsynaptic potentials (uEPSPs) in the PV-IN.

The inset shows an expanded view of the uEPSPs in the PV-IN generated from each AP in the excitatory cell.

( B–C ) Representative example traces of unitary excitatory postsynaptic currents (uEPSCs) generated in wild-type (WT) ( B ) and Kcnc1 -A421V/+ pairs of excitatory cells (top trace) and nearby PV-INs (bottom trace).

In response to a 20 Hz train of five APs generated in the excitatory cells, unitary inhibitory postsynaptic currents (uIPSCs) are recorded in the PV-INs (shown in gray), with the average of numerous sweeps shown for wild-type (WT) (black) or for Kcnc1 -A421V/+ (green).

( D ) Connection probability between the excitatory cell and the PV-IN for WT (n=9 of 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=6 of 43 pairs from N=5 mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) Connection probability between the excitatory cell and the PV-IN for WT (n=9 of 64 pairs from N=7 mice) and Kcnc1 -A421V/+ (n=6 of 43 pairs from N=5 mice).} a421v-kv31-membrane-trafficking-impaired⟧

( E ) Average failure rate for WT and Kcnc1 -A421V/+ neuron pairs relative to presynaptic stimulation frequency.

( F ) Average uEPSC magnitude in response to a train of five APs generated at 20 Hz in WT and Kcnc1 -A421V/+ neuron pairs.

( G ) Paired-pulse ratio of the second uEPSC to the first uEPSC in WT and Kcnc1 -A421V/+ mice.

(H) Average synaptic latency between peak of AP to peak of uEPSC in both WT and Kcnc1 -A421V/+ mice.

[panels detected: a, b, c, d, e, f, g, h] === Figure 7 === Figure 7. Adult (P32–42) Kcnc1 -A421V/+ mice exhibit altered parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated synaptic neurotransmission.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{[panels detected: a, b, c, d, e, f, g, h] === Figure 7 === Figure 7. Adult (P32–42) Kcnc1 -A421V/+ mice exhibit altered parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated synaptic neurotransmission.} pv-in-inhibitory-synapse-altered-adult⟧

( A ) Example presynaptic cortical PV-IN and postsynaptic excitatory neuron.

Arrowhead and inset display the unitary inhibitory postsynaptic potentials (uIPSPs) induced in the postsynaptic cell when the PV-IN generates action potentials (APs).

( B–C ) Example traces of unitary inhibitory postsynaptic currents (uIPSCs) in both adult wild-type (WT) ( B ) and Kcnc1 -A421V/+ ( C ) pairs of synaptically connected neurons. 10 APs were generated in the PV-IN at 40 Hz, and the evoked uIPSCs are displayed in the trace below where the black and green traces are the averages of numerous individual sweeps shown in gray.

( D ) Connection probability between WT (14 of 36, N=8 mice) and Kcnc1 -A421V/+ (13 of 36, N=8 mice) pairs PV-INs and nearby excitatory cells.⟦>zach claim=gap: @{( D ) Connection probability between WT (14 of 36, N=8 mice) and Kcnc1 -A421V/+ (13 of 36, N=8 mice) pairs PV-INs and nearby excitatory cells.} This panel carries the adult PV-IN to excitatory connection probability, which no claim in the tree records.⟧

( E ) Average frequency-dependent rate of failure for the first five APs in adult WT and Kcnc1 -A421V/+ neuron pairs.

(F–H) Average uIPSC magnitude of the first five APs for adult WT and Kcnc1 -A421V/+ at 20 Hz ( F ), 40 Hz ( G ), and 80 Hz ( H ).

(I–J) Paired-pulse ratios for WT and Kcnc1 -A421V/+ neuron pairs (uIPSC 2 /uIPSC 1 provided in I , uIPSC last /uIPSC first provided in J ) relative to stimulus frequency.

( K ) Average latency from AP peak to onset of the uIPSC in WT and Kcnc1 -A421V/+ mice.

Data are shown as mean ± SEM or individual values, and significance (*p<0.05, **p<0.01) was determined using unpaired t-test or repeated-measures two-way ANOVA.⟦>zach claim=bd91acf8-a01d-4713-8382-8a0757cbd86a: @{Data are shown as mean ± SEM or individual values, and significance (*p<0.05, **p<0.01) was determined using unpaired t-test or repeated-measures two-way ANOVA.} pv-in-inhibitory-synapse-altered-adult⟧

[panels detected: a, b, c, d, e, f, g, h, i, j, k] === Figure 8 === Figure 8. In vivo two-photon (2P) calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice.⟦>zach claim=2d6bb516-6c05-4a2f-bb64-9e9142bbf326: @{[panels detected: a, b, c, d, e, f, g, h, i, j, k] === Figure 8 === Figure 8. In vivo two-photon (2P) calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1 -A421V/+ mice.} in-vivo-hypersynchronous-discharges-mutant-only⟧

( A ) Experimental setup for in vivo 2P calcium imaging with representative calcium transients from cells expressing AAV-hSyn-GCaMP8m and mean dF/F 0 of the whole field of view (FOV) aligned to locomotion speed.

( B ) Representative 2P field of view during a hypersynchronous discharge in a Kcnc1 -A421V/+ mouse.

( C ) Mean dF/F of the field of view (top), calcium transients of individual somata (middle), and locomotion speed (bottom) during a paroxysmal discharge in the Kcnc1 -A421V/+ mouse relative to typical baseline activity shown in a wild-type (WT) mouse.

Note that, in contrast to epochs of low-amplitude synchronization in WT associated with transition from quiet wakefulness to locomotion, there is no locomotion during the larger-amplitude hypersynchronous discharges identified in Kcnc1 -A421V/+ mice.

( D ) Frequency of paroxysmal discharges in each mouse (N=5 WT, N = 7 Kcnc1 -A421V/+ mice).⟦>zach claim=ba388b6b-662d-4f09-b481-11f90f7057dc: @{( D ) Frequency of paroxysmal discharges in each mouse (N=5 WT, N = 7 Kcnc1 -A421V/+ mice).} a421v-kv31-membrane-trafficking-impaired⟧

( E ) Experimental design for in vivo 2P calcium imaging of somata positive (PV+) and negative (PV–) for parvalbumin.

( F ) Example calcium transients of PV+ (bottom) and PV– (top) somata aligned to locomotion speed in a WT (left) and Kcnc1 -A421V mouse (right).

( G–H ) Transients per minute during quiet rest in ( G ) PV– (WT, N=n=885 cells, 4 mice, mean = 1.17; Kcnc1 -A421V, n=1041 cells, N=3 mice, mean = 1.63) and ( H ) PV+ cells (WT, N=4 mice, n=110 cells, mean = 0.94; Kcnc1 -A421V, N=3 mice, n=100 cells, mean = 1.50).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{( G–H ) Transients per minute during quiet rest in ( G ) PV– (WT, N=n=885 cells, 4 mice, mean = 1.17; Kcnc1 -A421V, n=1041 cells, N=3 mice, mean = 1.63) and ( H ) PV+ cells (WT, N=4 mice, n=110 cells, mean = 0.94; Kcnc1 -A421V, N=3 mice, n=100 cells, mean = 1.50).} in-vivo-pv-minus-transient-frequency-increased⟧

( I–J ) Mean peak height in ( I ) PV– (WT, mean = 0.46; Kcnc1 -A421V, mean = 0.41) and ( J ) PV+ cells (WT, mean = 0.48; Kcnc1 -A421V , mean = 0.40).

Data points are shaded by mouse identity.

Statistical comparisons were performed using mixed-effects modeling.

[panels detected: a, b, c, d, e, f, g, h, i, j] === Figure 8s1 === Figure 8—figure supplement 1. Analysis of in vivo two-photon calcium imaging data.⟦>zach claim=no-assertion: @{[panels detected: a, b, c, d, e, f, g, h, i, j] === Figure 8s1 === Figure 8—figure supplement 1. Analysis of in vivo two-photon calcium imaging data.} A bare supplement title naming the analysis, with no finding asserted.⟧

( A ) PV– (wild-type [WT], n=847 cells, N=4 mice; Kcnc1 -A421V, n=901 cells, N=3 mice) and ( B ) PV+ cells (WT, n=65 cells; Kcnc1 -A421V, n=60 cells).⟦>zach claim=no-assertion: @{( A ) PV– (wild-type [WT], n=847 cells, N=4 mice; Kcnc1 -A421V, n=901 cells, N=3 mice) and ( B ) PV+ cells (WT, n=65 cells; Kcnc1 -A421V, n=60 cells).} A panel label giving only the cell and mouse counts for each group.⟧

( C–D ) Transients per minute during running in ( C ) PV– (WT, n=885 cells, N=4 mice; Kcnc1 -A421V, n=1041 cells, N=3 mice) and ( D ) PV+ cells (WT, n=110 cells, N=4 mice; Kcnc1 -A421V, n=100 cells, N=3 mice).⟦>zach claim=890a5223-4eb7-4dd0-a112-42e195398da1: @{( C–D ) Transients per minute during running in ( C ) PV– (WT, n=885 cells, N=4 mice; Kcnc1 -A421V, n=1041 cells, N=3 mice) and ( D ) PV+ cells (WT, n=110 cells, N=4 mice; Kcnc1 -A421V, n=100 cells, N=3 mice).} in-vivo-pv-minus-transient-frequency-increased⟧

( E–F ) Mean transient height in ( E ) PV– and ( F ) PV+ cells.

Error bars indicate standard error of the mean, and values next to data points are the mean by genotype.

Data points are shaded by mouse identity.

Statistical comparisons were performed using mixed-effects modeling.

( G–H ) Boxplot of percent of active ( G ) PV– and ( H ) PV+ cells for each mouse.

[panels detected: a, b, c, d, e, f, g, h] === Figure 9 === Figure 9. Kcnc1 -A421V/+ mice exhibit spontaneous seizures and seizure-induced death.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{[panels detected: a, b, c, d, e, f, g, h] === Figure 9 === Figure 9. Kcnc1 -A421V/+ mice exhibit spontaneous seizures and seizure-induced death.} spontaneous-seizures-and-sudep-kcnc1⟧

( A ) Representative example trace of the electroencephalogram (EEG) collected from an adult Kcnc1 -A421V/+ mouse during a nonfatal seizure.

After the seizure-related spike-wave discharges, there are large spikes that are associated with diffuse whole-body jerks.

( B ) Representative generalized tonic-clonic seizure resulting in seizure-induced sudden death in a Kcnc1 -A421V/+ mouse.

( C ) Raster plot indicating nonfatal seizures (blue bars), seizure-induced sudden death (red bars), interictal runs of spikes (green bars) without clear behavior manifestation, and periods of repetitive myoclonic seizures (yellow shading) for each mouse examined (N=12).⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{( C ) Raster plot indicating nonfatal seizures (blue bars), seizure-induced sudden death (red bars), interictal runs of spikes (green bars) without clear behavior manifestation, and periods of repetitive myoclonic seizures (yellow shading) for each mouse examined (N=12).} spontaneous-seizures-and-sudep-kcnc1 — This raster displays the per-mouse record of spontaneous seizures and seizure-induced deaths across the twelve mutants that the claim reports.⟧

Recordings in wild-type (WT) (N = 4) control mice did not show epileptic seizures or runs of spikes.⟦>zach claim=b066dc9f-dd75-43a8-9692-1005de9d2d30: @{Recordings in wild-type (WT) (N = 4) control mice did not show epileptic seizures or runs of spikes.} spontaneous-seizures-and-sudep-kcnc1 — The claim states that all four WT controls showed zero seizures on video-EEG.⟧

[panels detected: a, b, c]


## tables

Table 1. Membrane and action potential properties of WT and Kcnc1 -A421V/+ neurons at P16–21. AP, action potential; ADP, afterdepolarization; AHP, afterhyperpolarization; P, postnatal day; WT, wild type.⟦>zach claim=52b1301c-8ece-44c1-b25c-53e20013391d: @{Table 1. Membrane and action potential properties of WT and Kcnc1 -A421V/+ neurons at P16–21. AP, action potential; ADP, afterdepolarization; AHP, afterhyperpolarization; P, postnatal day; WT, wild type.} excitatory-neurons-unaffected-juvenile⟧

Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=20,9) –72.3±1.3 –39.8±0.8 253±8 –183±9 52.1±1.8 0.40±0.02 142±13 93±9 –63.7±1.0 Kcnc1 -A421V/+ (N=36, 12) –67.4±1.3 –41.6±0.5 252±9 –138±8 57.1±1.6 0.54±0.03 170±21 145±19 –63.0±0.8 Statistical comparison *p=0.017 p=0.065 p=0.96 **p=0.0012 p=0.055 **p=0.0053 p=0.29 p=0.19 p=0.62 Layer IV exc. cells WT (N=23, 3) –66.7±0.7 –43.4±0.7 303±13 –68.6±3.9 79.8±1.3 1.06±0.05 155±15 34±6 –60.2±0.5 Kcnc1 -A421V/+ (N=22,3) –67.6±1.0 –41.8±0.7 286±15 –64.5±3.7 77.8±1.6 1.10±0.04 159±14 37±6 –60.8±0.6 Statistical comparison p=0.49 p=0.11 p=0.38 p=0.45 p=0.35 p=0.46 p=0.86 p=0.69 p=0.47 Layer V PV-INs WT (N=15, 3) –65.9±1.2 –38.6±1.0 309±22 –223±17 57.5±1.9 0.37±0.02 147±13 85±11 –65.8±1.0 Kcnc1 -A421V/+ (N=12, 3) –66.3±1.7 –40.4±1.0 278±26 –164±15 59.0±2.1 0.47±0.03 141±17 97±13 –65.7±1.2 Statistical comparison p=0.82 p=0.21 p=0.38 *p=0.016 p=0.60 *p=0.014 p=0.76 p=0.49 p=0.92 RTN WT (N=18, 4) –55.1±1.4 –39.2±0.8 227±15 –189±11 49.2±1.8 0.38±0.02 247±39 24±7 –64.5±0.5 Kcnc1 -A421V/+ (N=19, 5) –57.5±2.3 –38.1±1.0 195±12 –160±7 45.6±1.7 0.42±0.02 236±30 48±10 –63.7±0.8 Statistical comparison p=0.39 p=0.41 p=0.10 *p=0.034 p=0.15 p=0.092 p=0.82 p=0.0805 p=0.40 Table 2. Membrane and AP properties of adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=20,9) –72.3±1.3 –39.8±0.8 253±8 –183±9 52.1±1.8 0.40±0.02 142±13 93±9 –63.7±1.0 Kcnc1 -A421V/+ (N=36, 12) –67.4±1.3 –41.6±0.5 252±9 –138±8 57.1±1.6 0.54±0.03 170±21 145±19 –63.0±0.8 Statistical comparison *p=0.017 p=0.065 p=0.96 **p=0.0012 p=0.055 **p=0.0053 p=0.29 p=0.19 p=0.62 Layer IV exc. cells WT (N=23, 3) –66.7±0.7 –43.4±0.7 303±13 –68.6±3.9 79.8±1.3 1.06±0.05 155±15 34±6 –60.2±0.5 Kcnc1 -A421V/+ (N=22,3) –67.6±1.0 –41.8±0.7 286±15 –64.5±3.7 77.8±1.6 1.10±0.04 159±14 37±6 –60.8±0.6 Statistical comparison p=0.49 p=0.11 p=0.38 p=0.45 p=0.35 p=0.46 p=0.86 p=0.69 p=0.47 Layer V PV-INs WT (N=15, 3) –65.9±1.2 –38.6±1.0 309±22 –223±17 57.5±1.9 0.37±0.02 147±13 85±11 –65.8±1.0 Kcnc1 -A421V/+ (N=12, 3) –66.3±1.7 –40.4±1.0 278±26 –164±15 59.0±2.1 0.47±0.03 141±17 97±13 –65.7±1.2 Statistical comparison p=0.82 p=0.21 p=0.38 *p=0.016 p=0.60 *p=0.014 p=0.76 p=0.49 p=0.92 RTN WT (N=18, 4) –55.1±1.4 –39.2±0.8 227±15 –189±11 49.2±1.8 0.38±0.02 247±39 24±7 –64.5±0.5 Kcnc1 -A421V/+ (N=19, 5) –57.5±2.3 –38.1±1.0 195±12 –160±7 45.6±1.7 0.42±0.02 236±30 48±10 –63.7±0.8 Statistical comparison p=0.39 p=0.41 p=0.10 *p=0.034 p=0.15 p=0.092 p=0.82 p=0.0805 p=0.40 Table 2. Membrane and AP properties of adult (P32–42) WT and Kcnc1 -A421V/+ PV-INs.} pv-in-ap-waveform-altered-downstroke-apd50⟧

AT, action potential; PV-INs, parvalbumin-positive fast-spiking GABAergic inhibitory interneurons; P, postnatal day; WT, wild type.

Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=14,3) –66.1±1.4 –41.5±0.9 294±25 –248±21 51.1±2.0 0.31±0.02 141±11 91±10 –67.6±1.1 Kcnc1 -A421V/+ (N=17, 5) –68.3±2.3 –43.1±0.6 324±16 –164±18 67.8±2.3 0.58±0.07 173±25 106±23 –66.0±1.2 Statistical comparison p=0.45 p=0.16 p=0.29 **p=0.0051 ***p<0.001 **p=0.0026 p=0.28 p=0.87 p=0.34 Layer IV exc. cells WT (N=12,4) –68.6±0.9 –41.7±0.5 363±17 –83.2±5.1 83.4±0.9 0.89±0.04 110±10 88±10 –55.7±1.1 Kcnc1 -A421V/+ (N=12, 4) –67.2±0.5 –40.0±0.7 338±18 –78.6±4.7 81.0±1.7 0.92±0.03 131±10 58±8 –56.8±0.9 Statistical comparison p=0.19 p=0.057 p=0.33 p=0.52 p=0.24 p=0.69 p=0.14 *p=0.023 p=0.47 Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent ( Mus musculis ) Kcnc1 -Flox(A421V) This study See Figure 1 Genetic reagent ( Mus musculis ) B6-Tg(Pvalb-tdTomato)15Gfng/J JAX RRID: IMSR_JAX:027395 Referred to as Pvalb-tdT Genetic reagent ( Mus musculis ) FVB/N – Tmem163 Tg(ACTB-cre)2Mrt /J JAX RRID: IMSR_JAX:003376 Referred to as ActB-Cre Genetic reagent ( Mus musculus ) C57BL/6J JAX RRID: IMSR_JAX:000664 Recombinant DNA reagent AAV9-syn-jGCaMP8m-WPRE Addgene #162375 RRID: Addgene_162375 Diluted to a titer of 2e12 in sterile PBS. 60 nL delivered Recombinant DNA reagent PHP.eB-E6-S5E2-dTom-nlsdTom Addgene #135630 RRID: Addgene_135630 Diluted to a titer of 2e12 in sterile PBS. 60 nL delivered Software pClamp ClampFit 11.2 RRID: SCR_011323 Software MATLAB MathWorks RRID: SCR_001622 Software NeuroScore (EEG Analysis) Data Sciences International Software Analysis of whole-cell electrophysiology This paper; Wengert et al., 2021 Software Analysis of whole-cell electrophysiology This paper; Wengert et al., 2021 https://doi.org/10.12751/g-node.bqni9h Software Analysis of two-photon imaging This paper; Goff et al., 2023 https://doi.org/10.12751/g-node.bqni9 Antibody Anti-Kv3.1b (rabbit polyclonal) Alomone Labs Cat# APC-014 RRID: AB_2040166 1: 500 dilution Antibody Anti-Parvalbumin (mouse monoclonal) Millipore Cat# MAB1572 RRID: AB_2174013 1:1000 Antibody Anti-rabbit-Alexa Fluor 488 (goat) Molecular Probes Cat# A11034 RRID: AB_2576217 1:1000 Antibody Anti-mouse-Alexa Fluor 568 (goat) Molecular Probes Cat# A21124 RRID: AB_141611 1:1000 Cell Line ( Homo sapiens ) HEK-293T Cells ATCC, CRL-3216 RRID: CVCL_0063 Recombinant DNA reagent cDNA plasmid for human KCNC1 Clatot et al., 2023 Reference sequence NM_001112741.2 Available upon request Other DAPI Thermo Fisher Scientific Cat# D1306 RRID: AB_2629482 1:50,000⟦>zach claim=04db8b33-39ce-43b1-8df4-63a156fbc874: @{Cell type Group V m (mV) AP threshold (mV) Upstroke velocity (mV/ms) Downstroke velocity (mV/ms) AP amplitude (mV) APD50 (ms) Input resistance (MΩ) Rheobase (pA) AHP (mV) Layer II-IV PV-INs WT (N=14,3) –66.1±1.4 –41.5±0.9 294±25 –248±21 51.1±2.0 0.31±0.02 141±11 91±10 –67.6±1.1 Kcnc1 -A421V/+ (N=17, 5) –68.3±2.3 –43.1±0.6 324±16 –164±18 67.8±2.3 0.58±0.07 173±25 106±23 –66.0±1.2 Statistical comparison p=0.45 p=0.16 p=0.29 **p=0.0051 ***p<0.001 **p=0.0026 p=0.28 p=0.87 p=0.34 Layer IV exc. cells WT (N=12,4) –68.6±0.9 –41.7±0.5 363±17 –83.2±5.1 83.4±0.9 0.89±0.04 110±10 88±10 –55.7±1.1 Kcnc1 -A421V/+ (N=12, 4) –67.2±0.5 –40.0±0.7 338±18 –78.6±4.7 81.0±1.7 0.92±0.03 131±10 58±8 –56.8±0.9 Statistical comparison p=0.19 p=0.057 p=0.33 p=0.52 p=0.24 p=0.69 p=0.14 *p=0.023 p=0.47 Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent ( Mus musculis ) Kcnc1 -Flox(A421V) This study See Figure 1 Genetic reagent ( Mus musculis ) B6-Tg(Pvalb-tdTomato)15Gfng/J JAX RRID: IMSR_JAX:027395 Referred to as Pvalb-tdT Genetic reagent ( Mus musculis ) FVB/N – Tmem163 Tg(ACTB-cre)2Mrt /J JAX RRID: IMSR_JAX:003376 Referred to as ActB-Cre Genetic reagent ( Mus musculus ) C57BL/6J JAX RRID: IMSR_JAX:000664 Recombinant DNA reagent AAV9-syn-jGCaMP8m-WPRE Addgene #162375 RRID: Addgene_162375 Diluted to a titer of 2e12 in sterile PBS. 60 nL delivered Recombinant DNA reagent PHP.eB-E6-S5E2-dTom-nlsdTom Addgene #135630 RRID: Addgene_135630 Diluted to a titer of 2e12 in sterile PBS. 60 nL delivered Software pClamp ClampFit 11.2 RRID: SCR_011323 Software MATLAB MathWorks RRID: SCR_001622 Software NeuroScore (EEG Analysis) Data Sciences International Software Analysis of whole-cell electrophysiology This paper; Wengert et al., 2021 Software Analysis of whole-cell electrophysiology This paper; Wengert et al., 2021 https://doi.org/10.12751/g-node.bqni9h Software Analysis of two-photon imaging This paper; Goff et al., 2023 https://doi.org/10.12751/g-node.bqni9 Antibody Anti-Kv3.1b (rabbit polyclonal) Alomone Labs Cat# APC-014 RRID: AB_2040166 1: 500 dilution Antibody Anti-Parvalbumin (mouse monoclonal) Millipore Cat# MAB1572 RRID: AB_2174013 1:1000 Antibody Anti-rabbit-Alexa Fluor 488 (goat) Molecular Probes Cat# A11034 RRID: AB_2576217 1:1000 Antibody Anti-mouse-Alexa Fluor 568 (goat) Molecular Probes Cat# A21124 RRID: AB_141611 1:1000 Cell Line ( Homo sapiens ) HEK-293T Cells ATCC, CRL-3216 RRID: CVCL_0063 Recombinant DNA reagent cDNA plasmid for human KCNC1 Clatot et al., 2023 Reference sequence NM_001112741.2 Available upon request Other DAPI Thermo Fisher Scientific Cat# D1306 RRID: AB_2629482 1:50,000} pv-in-ap-waveform-altered-downstroke-apd50⟧