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

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