*4.7. Immunostaining*

Mice were fixed with 4% PFA and brain sections were cut into 24-well plates. They were then blocked with 10% horse serum at RT for 1 h and then incubated with primary antibodies at 4 ◦C overnight. After washing, sections were incubated with anti-mouse-555 and anti-rabbit-488 secondary antibodies at RT for 2 h. After washing, cells were then mounted on slides with anti-fade mounting medium containing <sup>4</sup>,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA, P36962) and imaged using a Zeiss (Oberkochen, Germany) 710 laser scanning confocal microscope equipped with a 63 × objective.

#### *4.8. Acute Hippocampal Slice Preparation*

Adult male and female (5–6 weeks old) mice were used for all acute slice electrophysiological recordings. Mice were anesthetized with isoflurane and decapitated. Brains were rapidly removed and washed with ice-cold sucrose cutting solution. The sucrose solution was made up of the following (in mM): 60 NaCl, 3 KCl, 28 NaHCO3, 1.25 NaH2PO4, 7.5 Glucose, 0.5 CaCl2, 4.5 MgCl2. Brain hemispheres were dissected and mounted following a 45◦ cut of the dorsal cerebral hemisphere(s). Modified transverse slices (300 μm) were made by a Leica (Wetzlar, Germany, VT1200S) vibrating microtome in ice-cold sucrose that was continuously bubbled with carbogen (95% O2/5% CO2). Slices were recovered at 32 ◦C in sucrose solution for 15 min, at which time the solution temperature was slowly lowered to room temperature where it remained for the remainder of the recording day. Slices were exposed to pharmacological treatments (treatment or vehicle) during the slicing procedure and in recovery.

#### *4.9. Whole-Cell Current Clamp Recordings*

Following a 2-h recovery in sucrose cutting solution with or without pharmacological treatment (SB 203580 5 μM and 0.1% DMSO vehicle, respectively), hippocampal slices were transferred to a recording chamber submerged in artificial cerebral spinal fluid (ACSF) with the temperature maintained at 33 ◦C (±1 ◦C). The ACSF consisted of the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 2 CaCl2, (pH 7.4). In select recordings, 5 μM SB 203580 was added to the bath solution. The recording chamber was continuously perfused with carbogen-bubbled ACSF at a rate of 3 mL/min. Somatic whole-cell patch clamp recordings were performed on identified somata of hippocampal CA1 pyramidal neurons, which were viewed using infrared di fferential interference contrast (DIC) on an upright Zeiss (Oberkochen, Germany) Examiner. Cells were patched with 4–5 M Ω borosilicate glass pipettes pulled from a Narishige (Amityville, NY, USA) vertical puller and filled with K<sup>+</sup> gluconate-based intracellular solution consisting of the following (in mM): 20 KCl, 130 K-gluconate, 2 MgCl, 0.1 EGTA, 2 Na2ATP, 0.3 NaGTP, 10 HEPES, 10 Phosphocreatine with pH adjusted with KOH, and HCl to a final value of 7.25–7.30 and an osmolarity of 290–300 mOsm.

AP firing properties were measured from whole-cell recordings in current clamp mode in the conditions described above. All data were recorded with a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA, USA) and a Digidata 1440A digitizer. Signals were low-pass filtered at 5 kHz and digitized at 10 kHz using Clampex 10.7 software and were acquired in bridge balance mode to compensate for series resistance. Liquid junction potential was not corrected for. Passive membrane properties were measured after initial break-in in order to avoid dialysis as a result of solution exchange. Whole-cell capacitance and series resistance were measured from a Multiclamp 700B commander (Molecular Devices, San Jose, CA, USA). A voltage step of −10 mV was used, and the decay tau of the whole-cell capacitive transient current was used to calculate these parameters. Recordings where series resistance exceeded 25 M Ω or resting membrane potential was more depolarized than −55 mV were discarded. Input resistance was calculated as the slope of the current-voltage (I-V) curve in response to current steps from −50 to 50 pA in 50 pA steps (three steps in total). To evoke action potentials in patched CA1 pyramidal neurons, square 500 ms current pulses were elicited in 50 pA steps with current injections ranging from −200 pA to +300 pA. Two sweeps at each magnitude were elicited and the average response of these sweeps was used for each cell. All measures of action potential waveform were taken from the first spike in response to a 150 pA injection and inter-spike measurements, including inter-spike interval, were recorded between the first two spikes in a train elicited by a 150 pA square current. This current magnitude was used as it was the minimum current that provoked AP firing in 100% of cells patched. Additionally, second-long ramp current injections were elicited at 400 pA/sec and the latency to fire was calculated as the time from the initiation of the current injection to AP threshold (first AP). AP threshold and rheobase were measured from ramp injections, with rheobase recorded as the current magnitude required to produce the first AP (at threshold voltage) in the ramp.

#### *4.10. Seizure Behavioral Assays*

Kainic acid (Sigma, St. Louis, MO, USA, K0250) was administered i.p. at a dose of 25 mg/kg. Animals were monitored for 60 min after the injection. Behavioral responses were recorded using a video camera and scored using the following: stage 0, normal behavior; stage 1, immobility and rigidity; stage 2, head bobbing; stage 3, forelimb clonus and rearing; stage 4, continuous rearing and falling; stage 5, clonic–tonic seizure; stage 6, death (Racine, 1972). Total seizure scores were calculated by summing up every five-minute score. The behavioral assessments described above were performed in a blind manner.

## *4.11. Statistical Analysis*

Biochemistry and behavior data were analyzed by Origin 2018b (Northampton, MA, USA) by two-tailed Student's *t*-test and two-way ANOVA, respectively. Electrophysiological data were analyzed by GraphPad Prism 7 (San Diego, CA, USA, 7.0 d). For all electrophysiological experiments, the experimenter was blinded to the genotypes. For analysis of the pharmacological impact on single AP parameters and in response to ramp current injections in WT and Kv4.2TA slices, a one-way ANOVA (ordinary), or one-way ANOVA on ranks (Kruskal–Wallis test) was used and was corrected for multiple comparisons with Dunnett's test (ordinary) or Dunn's test (ranks). The use of parametric or non-parametric analysis was determined after testing for normal distribution of the data using the D'Agostino and Pearson normality test (alpha level = 0.05). Non-parametric statistics were used if the data failed normality testing. For all analysis of firing frequency in response to sequential current steps, a two-way ANOVA with Tukey's post hoc test was used. All the data are presented as mean ± SEM.

**Author Contributions:** J.-h.H., C.M., and D.A.H. conceived and planned the experiments; J.-h.H. and C.M. carried out the experiments; J.-h.H. and C.M. analyzed and interpreted the data; J.-h.H., C.M., and D.A.H. drafted the paper; All authors provided critical feedback and helped shape the research, analysis, and paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank Vincent Schram at the NICHD imaging core facility for helping with imaging and members of the Ho ffman lab for advice and suggestions. This work was supported by the *Eunice Kennedy Shriver* NICHD Intramural Research Program.

**Conflicts of Interest:** The authors state no competing interests.
