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. 2023 Aug 8;8(15):e170399.
doi: 10.1172/jci.insight.170399.

A hyperthermic seizure unleashes a surge of spreading depolarizations in Scn1a-deficient mice

Isamu Aiba, Yao Ning, Jeffrey L Noebels

A hyperthermic seizure unleashes a surge of spreading depolarizations in Scn1a-deficient mice

Isamu Aiba et al. JCI Insight. .

Abstract

Spreading depolarization (SD) is a massive wave of cellular depolarization that slowly migrates across the brain gray matter. Cortical SD is frequently generated following brain injury, while less is understood about its potential contribution to genetic disorders of hyperexcitability, such as SCN1A-deficient epilepsy, in which febrile seizure often contributes to disease initiation. Here we report that spontaneous SD waves are predominant EEG abnormalities in the Scn1a-deficient mouse (Scn1a+/R1407X) and undergo sustained intensification following a single hyperthermic seizure. Chronic DC-band EEG recording detected spontaneous SDs, seizures, and seizure-SD complexes in Scn1a+/R1407X mice but not WT littermates. The SD events were infrequent, while a single hyperthermia-induced seizure robustly increased SD frequency over 4-fold during the initial postictal week. This prolonged neurological aftermath could be suppressed by memantine administration. Video, electromyogram, and EEG spectral analysis revealed distinct neurobehavioral patterns; individual seizures were associated with increased motor activities, while SDs were generally associated with immobility. We also identified a stereotypic SD prodrome, detectable over a minute before the onset of the DC potential shift, characterized by increased motor activity and bilateral EEG frequency changes. Our study suggests that cortical SD is a pathological manifestation in SCN1A-deficient epileptic encephalopathy.

Keywords: Epilepsy; Neuroscience.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Seizure and SD phenotype of Scn1a+/RX mice before and after a hyperthermic seizure.
(A) Electrode positions: from the top, #1 right anterior, #2 left anterior, #3 right posterior, #4 left posterior. (B) Compressed trace showing a 24-hour recording. SDs are reliably detected as sharp negative shift over stable baseline. (C–E) Expanded representative traces of SD, seizure, and seizure+SD complex. (F–H) Hyperthermic seizure robustly increased SD and seizure incidence. (F) Raster plots of seizure and SD incidence in WT and Scn1a+/RX mice. WT mice had no seizure or SD. Three mice exclusively had seizures (“seizure-only”). Seven mice died or became moribund during the study. The same Scn1a+/RX event data are presented in a cumulative histogram (G) and pie chart (H) showing proportion of seizure, SD, and seizure+SD events during baseline and after a hyperthermic seizure in Scn1a+/RX mice that survived the recording period, excluding the “seizure-only” mice. (I) Quantification of event frequencies. Frequencies of SD and total events were increased after a hyperthermic seizure. “Seizure-only” mice were excluded from this analysis. Two-way ANOVA and post hoc Tukey’s test. (J) Chronological analysis of SD, seizure, and seizure+SD events.
Figure 2
Figure 2. SD generation during hyperthermic seizures induced with a heating lamp in Scn1a+/RX mice.
(A and B) Representative EEG showing seizure and SD. Top: DC; middle: high pass (>1 Hz); bottom: power spectrum of EEG (anterior electrode). (C) Postictal SD was less common in WT mice: 77% (10/13) of Scn1a+/RX mice developed SD following seizure, while 27% (3/11) of WT mice did so. (D) Consistent with previous studies, Scn1a+/RX mice showed a lowered thermal threshold for seizure. WT: n = 11; Scn1a+/RX: n = 13; P = 0.007, Mann-Whitney U test. **P < 0.01.
Figure 3
Figure 3. Electrophysiological characteristics of PGES and postictal SD.
(A) Representative trace showing the temporal sequence of PGES (depressed EEG amplitude, blue window) and postictal SD generation. Top: DC; middle: high pass (>1 Hz); bottom: EEG converted into power. (B and C) The PGES incidence (B) and duration (C) were similar in seizure without postictal SD and seizure with postictal SD. Seizure only: n = 55; seizure+SD: n = 20. (D) The latency to SD after seizure termination is significantly prolonged after a hyperthermic seizure. Baseline: n = 11; after hyperthermic seizure: n = 32. (E–H) Comparison of seizure/SD kinetics between those in isolated events and those in the seizure+SD complex. The duration of SD in the seizure+SD complex is shorter than the duration of SD detected alone (E), while the DC amplitudes were not different (F). Similarly, the duration of seizure in the seizure+SD complex is shorter than the duration of seizure that appeared without SD (G), while the DC amplitudes were not different (H). SD only: n = 160; seizure only: n = 95; seizure+SD: n = 43. Statistics were computed by Mann-Whitney U test.
Figure 4
Figure 4. Prolonged memantine administration inhibits the hyperthermic seizure–induced exacerbation of depolarizing events in Scn1a+/RX mice.
Cumulative histogram bars show SD incidence (orange), seizures (blue), and seizure+SD complexes (red) before and after hyperthermic seizure. Box plots show total event frequency (total events per hour) during baseline (white) and after hyperthermic seizure (red). (A) Pattern of events in saline-pretreated control Scn1a mutants. (B–D) Efficacy of single-dose memantine (10 mg/kg, i.p.) pretreatment administered 30–60 minutes before hyperthermic seizure (B), efficacy of memantine after treatment repeated 6 and 12 hours after hyperthermic seizure (C), and combined pre- and posttreatment data (D) were analyzed. The duration of treatment is shown in the yellow shade, and the duration of the posthyperthermic seizure period in the pink shade. At right, the frequency of total events during baseline and following the hyperthermic seizure is shown. In each group, n = 8 mice. Statistics calculated by paired Wilcoxon’s signed-rank test.
Figure 5
Figure 5. Subconvulsive PTZ stimulation partially mimics hyperthermic seizure effect.
(A) Representative traces of EEG activity following PTZ injection (30 mg/kg, i.p.) in 7 Scn1a+/RX mice. PTZ increased interictal epileptic discharges for 30 minutes without seizure in this mouse. Traces from top: right anterior, left anterior, right posterior, left posterior. (B) Raster plots of SD, seizure, and seizure+SD complex during baseline and after PTZ injection (pink shade). Two mice died during the recording. (C) The same results presented in cumulative histogram of SD, seizure, and seizure+SD incidences. (D) Quantitative comparison of frequencies of SD, seizure, seizure+SD, and total events before and after PTZ injection. n = 5; statistics calculated by paired Wilcoxon’s signed-rank test.
Figure 6
Figure 6. Analysis of EMG motor activity associated with SD, seizure, and seizure+SD.
Top: Unilateral anterior and posterior EEG and simultaneous neck EMG trace. Middle: Raster plot of individual analyzed EMG signal patterns. Each lane represents a single event in a representative animal. Bottom: Averaged traces of EMG activity are presented as mean ± SEM. (A) Seizure is associated with an abrupt increase in EMG signal, which is followed by motor activations. (B) SD is associated with prodromal behavior activation, followed by suppression as DC shift is detected in the frontal cortex. (C) Seizure+SD is also associated with initial convulsive motor activity, which is inhibited once SD is detected over the frontal cortex. Some motor activity is present after postictal SD but is reduced in comparison with seizure alone. n = 11, 47, and 9 events for seizure, SD, and seizure+SD, respectively. P < 0.001 in EMG patterns between events, aligned rank transformation ANOVA.
Figure 7
Figure 7. Locomotion changes associated with seizure, SD, and seizure+SD were analyzed using video images.
(A) Average traces (top, mean ± SEM) and raster plots (bottom; each lane shows a single event) 30 minutes before and after event onset (line at t = 0). n = 37, 67, and 14 events for seizure, SD, and seizure+SD, respectively. (B and C) Locomotion was also analyzed by a binary method (see Results). Comparison of locomotion 10 or 5 minutes before (B) and after (C) each event. Seizure+SD is associated with reduced pre- and postevent locomotion activity. (D–F) Comparisons of locomotion changes in individual events 3 minutes before and after each event. Seizure and SD did not show consistent directional changes; however, seizure+SD events consistently reduced locomotion activity. Two-way ANOVA; event: F = 2.01, P = 0.14; time: F = 15.93, P < 0.001; interaction: F = 1.23, P = 0.30.
Figure 8
Figure 8. Prodromal EEG frequency change precedes the onset of the negative DC potential shift of SD.
(A) DC and EEG power changes in isolated SD. EEG activities showed a robust high-frequency shift (yellow shade) more than a minute before the onset of the negative DC potential shift of SD. Note that complex EEG spikes are always detected at the onset of prodromal changes. DC, low-band (0–30 Hz, black), and high-band (30–120 Hz, red) EEG power in the SD-affected (top) and the contralateral hemisphere (bottom) are shown. (B) DC and EEG power changes during seizure+SD complex. (C) Quantification of the EEG power during baseline and prodromal phase from 45 isolated SDs. The EEG frequency was altered in both SD-affected and contralateral hemispheres (time effect: P < 0.0001 both hemispheres, repeated-measures ANOVA). (D) Same analysis of prodromal EEG frequency change in 21 seizure+SD complexes (time effect: P < 0.001 both hemispheres, repeated-measures ANOVA). *P < 0.05, ****P < 0.001, post hoc paired t test with Holm’s correction.
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