Impaired excitability of fast-spiking neurons in a novel mouse model of KCNC1 epileptic encephalopathy
- Division of Neurology, Department of Pediatrics, The Children’s Hospital of Philadelphia, United States
- Department of Neuroscience, The University of Pennsylvania Perelman School of Medicine, United States
- The Medical Scientist Training Program, The University of Pennsylvania Perelman School of Medicine, United States
- The Center for Brain Research in Development, Genetics, and Engineering (BRIDGE), United States
- The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, United States
- Department of Pathology & Laboratory Medicine, The University of Pennsylvania Perelman School of Medicine; The University of Pennsylvania, United States
- School of Arts and Sciences, The University of Pennsylvania Perelman School of Medicine, United States
- The Epilepsy Neurogenetics Initiative, The Children’s Hospital of Philadelphia, United States
- School of Engineering and Applied Sciences, The University of Pennsylvania Perelman School of Medicine, United States
- Department of Neurology, The University of Pennsylvania Perelman School of Medicine, United States
Figures
Figure 1 with 2 supplements
Design of a novel mouse model of KCNC1 developmental and epileptic encephalopathy.
(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.
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.
(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 (mm2) are not different between WT and Kcnc1-A421V/+ mice (N=3 mice/genotype). (C) Sensitivity rate (probability of a cell being tdTomato+ if it is parvalbumin+; N=6 mice/genotype). (D) False-positive probability (proportion of all tdTomato+ cells that are parvalbumin negative; N=6 mice/genotype).
Figure 1—figure supplement 2
Early postnatal development of Kcnc1-A421V/+ mice.
(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). 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. (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.
Figure 2
Impaired cognitive function in Kcnc1-A421V/+ mice.
(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. (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. (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.
Figure 3 with 1 supplement
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.
(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). (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). 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.
(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.
Figure 4 with 3 supplements
Impaired parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN) intrinsic excitability in juvenile and adult Kcnc1-A421V/+ mice.
(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). (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). (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.
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.
(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. 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.
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/+.
(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. (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.
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.
(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). (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. Data are shown as mean ± SEM, and significance (***p<0.001) was determined using repeated-measures two-way ANOVA.
Figure 5 with 1 supplement
Unaltered potassium currents and physiological function of excitatory neurons in postnatal day (P)16–21 Kcnc1-A421V/+ mice.
(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. (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). 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.
(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. Data are shown as mean ± SEM, and all results failed to reach significance determined via repeated-measures two-way ANOVA.
Figure 6 with 1 supplement
Juvenile (P16–21) Kcnc1-A421V/+ mice exhibit normal parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated inhibitory synaptic neurotransmission.
(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). (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.
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.
(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). (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.
Figure 7
Adult (P32–42) Kcnc1-A421V/+ mice exhibit altered parvalbumin-positive fast-spiking GABAergic inhibitory interneuron (PV-IN)-mediated synaptic neurotransmission.
(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. (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 (uIPSC2/uIPSC1 provided in I, uIPSClast/uIPSCfirst 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.
Figure 8 with 1 supplement
In vivo two-photon (2P) calcium imaging reveals paroxysmal hypersynchronous discharges and altered neuronal excitability in Kcnc1-A421V/+ mice.
(A) Experimental setup for in vivo 2P calcium imaging with representative calcium transients from cells expressing AAV-hSyn-GCaMP8m and mean dF/F0 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). (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). (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.
(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). (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). (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.
Figure 9
Kcnc1-A421V/+ mice exhibit spontaneous seizures and seizure-induced death.
(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). Recordings in wild-type (WT) (N=4) control mice did not show epileptic seizures or runs of spikes.
Author response image 1
Immunostaining of Kv3.
1 and Kv3.2 in sagittal mouse brain sections. (a) An example of intracellular Kv3.2 immunostaining signal, variable across the cortex of a WT mice independent of Kv3.1 expression (b) Kv3.2 is detectable intracellularly in most of the cells in the top panel but barely detectable in the lowest panel. (c) Representative image of Kv3.2 immunostaining signal in other sagittal mouse brain sections.
Videos
Video 1
Example in vivo two-photon (2P) calcium imaging of synchronous discharge in a Kcnc1-A421V/+ mouse.
Video 2
Example seizure with myoclonic jerks.
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.
| Cell type | Group | Vm (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 |
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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.
AT, action potential; PV-INs, parvalbumin-positive fast-spiking GABAergic inhibitory interneurons; P, postnatal day; WT, wild type.
| Cell type | Group | Vm (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.
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 – Tmem163Tg(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 |
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Impaired excitability of fast-spiking neurons in a novel mouse model of KCNC1 epileptic encephalopathy
eLife 13:RP103784.
https://doi.org/10.7554/eLife.103784.3
