Circumstances of SUDEP in DS mice.
To the best of our knowledge, there are no detailed descriptions of the circumstances of SUDEP in DS patients because these tragic events usually occur unexpectedly and unwitnessed (6
). Our previous studies have shown that many of our DS mice die in their fourth postnatal week of life (22
). To examine the circumstances of these sporadic deaths, continuous video recordings of 39 DS mice in their home cages were made from P25 to P28. During this monitoring period, 18 mice died and review of the video records revealed that all the deaths occurred immediately following a Racine 5 generalized tonic-clonic seizure. Analyses of the data indicated that in the 24 hours prior to death, the incidence of convulsive seizures was higher in the mice that died than in those that survived (Figure A). However, the Racine score of each seizure was 5.0, and durations of these convulsions were not statistically different between the 2 groups of mice (Figure A). These findings indicate that SUDEP is a seizure-induced event and that high incidence of Racine 5 seizures predicts SUDEP susceptibility in these mice, whereas seizure duration does not.
Spontaneous seizures and sudden unexpected deaths in DS mice.
Correlation between epilepsy and premature death in DS mice.
To examine the long-term relationship between epilepsy phenotype and SUDEP, 2-week video records of 17 DS mice were obtained during P20–P34. Consistent with our previous findings (22
), spontaneous seizures began at P20–P21. Of 17 mice monitored, 6 (35.3%) died during these recordings. A Kaplan-Meier plot of deaths reveals a correlation of premature death with seizure incidence in postnatal week 4 (P21–P28; Figure B), and all of the deaths occurred during the period of highest seizure incidence, P23–P27 (Figure B). The elevated cumulative incidence of seizures during postnatal week 4 corresponded to the period of high SUDEP susceptibility, and surviving mice had a much lower frequency of seizures than those that died prematurely (Figure C). Status epilepticus, defined as a single seizure or series of recurring seizures lasting more than 30 minutes (26
), is an exclusion criterion for the definition of SUDEP in patients (27
). In our recordings, 3 mice with status epilepticus were identified and excluded from SUDEP analyses. These mice had numerous recurring brief convulsions: 1.5 seizure/min in the first 30 minutes and 0.4 seizure/min in the following hour. All 3 mice survived the status epilepticus but died 8 ± 3 hours later. Together, these findings indicate a strong correlation between seizure history and deaths in DS mice.
Heart rate in DS, Dlx-Cre, and Mer-Cre mice.
1.1 channels are expressed in mouse brain and heart, and they play critical roles in the function of these organs (14
). To investigate whether global heterozygous KO of NaV
1.1 channels in DS mice causes cardiac defects that may predispose to SUDEP, we studied DS mice, heterozygous F/+
mice in which NaV
1.1 channels are selectively deleted in forebrain GABAergic interneurons (25
), and heterozygous F/+
mice in which NaV
1.1 channels are selectively deleted in the heart (Methods). Extensive immunocytochemical studies with specific antibodies demonstrated up to 50% reduced expression of NaV
1.1 channels in GABAergic interneurons in many regions of the cerebral cortex and hippocampus in F/+
mice, but no reduction in excitatory neurons in these brain regions (25
). Similarly, we found that F/+
mice have reduced expression of NaV
1.1 channels in the ventricles of the heart (55% of control; Supplemental Figure 1; supplemental material available online with this article; doi:
Continuous 8-hour video-EEG-ECG records were obtained from conscious unrestrained DS, F/+:Dlx-Cre+, F/+:MCre+, and WT mice from 8:00 am to 4:00 pm. Assessment of the resting heart rates indicated that DS and F/+:Dlx-Cre+ mice have slightly elevated, but not significantly different, resting heart rates compared with control mice (Figure , A and B, and Supplemental Table 1). F/+:MCre+ mice have significantly suppressed resting heart rate. These findings indicate that global heterozygous KO of NaV1.1 and forebrain interneuron-specific heterozygous KO of this channel has little or no effect on resting heart rate, whereas cardiac-specific heterozygous KO of these channels has a suppressive effect on resting heart rate.
Cardiovascular effects of global or conditional KO of Scn1a.
Heart rate is controlled primarily by input from the parasympathetic and sympathetic nervous systems to the sino-atrial node of the heart. To examine whether Scn1a KO in DS mice altered resting parasympathetic or sympathetic tone, the effects of pharmacological blockade of each of these branches of the autonomic nervous system were assessed. To differentiate effects due to brain-specific versus cardiac-specific gene KO, similar pharmacological tests were performed in F/+:Dlx-Cre+ and F/+:MCre+ mice and their respective control groups. Administration of a saturating dose of atropine, a muscarinic receptor antagonist that blocks parasympathetic signaling, caused an increase in heart rate in DS, F/+:Dlx-Cre+, and F/+:MCre+ mice similar to that found in their respective controls (Figure C and Supplemental Table 1). Administration of a saturating dose of propranolol, a β adrenergic receptor antagonist that blocks sympathetic signaling, caused a reduction of heart rate in DS, F/+:Dlx-Cre+, and F/+:MCre+ mice similar to that found in the corresponding controls (Figure C and Supplemental Table 1). Simultaneous administration of atropine and propranolol also produced decreases in DS, F/+:Dlx-Cre+, and F/+:MCre+ mice similar to those found in controls (Figure C and Supplemental Table 1). These findings indicate no detectable change in resting autonomic tone or intrinsic heart rate in Scn1a mutant mice.
Heart-rate variability in DS, Dlx-Cre, and Mer-Cre mice.
A decrease in heart-rate variability has been associated with higher mortality and an increased incidence of sudden cardiac death (29
). It has been observed in some patients with epilepsy and proposed as a risk factor for SUDEP (30
). Analyses of heart-rate variability of Scn1a
-KO mice showed that the coefficient of variation of resting heart rate was decreased for DS mice compared with WT and for F/+
mice compared with F/+
(Figure D). In contrast, an increase in the coefficient of variation of heart rate was observed for F/+
compared with F/+
mice (Figure D and Supplemental Table 2). These results indicate that heterozygous deletion of NaV
1.1 channels in all cells or only in forebrain GABAergic neurons decreases heart-rate variability, whereas heterozygous deletion in cardiac myocytes increases heart-rate variability.
To further investigate whether DS mice have suppressed heart-rate variability, we analyzed the characteristic intervals in the electrocardiogram. The SD of normal R-R intervals (SD NN), the SD of δ NN, and the root mean square of differences between adjacent normal R-R intervals (RMSSD) were all reduced in DS mice compared with WT mice (Figure , E–G, and Supplemental Table 2). These findings demonstrate that global heterozygous deletion of Scn1a suppresses heart-rate variability in DS mice. Like DS mice, F/+:Dlx-Cre+ mice had reduced heart-rate variability compared with controls when assessed from measurements of these ECG parameters (Figure , E–G, and Supplemental Table 2). In contrast, in F/+:MCre+ mice, all the indices of heart-rate variability were elevated (Figure , E–G, and Supplemental Table 2). These findings indicate that global or forebrain interneuron restricted heterozygous deletion of NaV1.1 channels reduced heart-rate variability, as detected by 4 separate measurements, whereas cardiac-specific heterozygous deletion tends to increase heart-rate variability.
Interictal atrioventricular block in DS mice.
The different intervals in the ECG provide a quantitative measure of action potential conduction in the different regions of the heart. No differences were observed in PR, QRS, QT, and corrected QT (QTc) intervals between genotypes, except for a small increase in PR interval in F/+:MCre+ mice (Supplemental Table 3). Thus, specific heterozygous deletion of Scn1a in forebrain interneurons does not influence the rate of cardiac conduction, whereas cardiac-specific heterozygous deletion of the gene leads to a small increase in PR interval. Further analysis of the resting ECG records showed a striking increase in the frequency of atrioventricular (AV) block (Figure ). Transient AV block is common in WT mice, but the frequency was higher in DS mice (2.24 ± 1.2 AV blocks/h) and F/+:Dlx-Cre+ mice (1.5 ± 0.2 AV blocks/h) compared with their corresponding controls (WT: 0.6 ± 0.4 and F/+:MCre–: 0.7 ± 0.5 AV blocks/h; P = 0.001 and 0.02; Figure ). In F/+:MCre+ mice, the frequency of AV blocks was slightly elevated (0.8 ± 0.5 AV blocks/h), but not significantly different from control (0.7 ± 0.6 AV blocks/h; P > 0.05; Figure ). These findings indicate that global and interneuron-specific heterozygous KO of Scn1a cause a significant increase in the frequency of AV block in DS mice, but targeted cardiac deletion of the gene does not.
Arrhythmias in DS mice during thermal seizure induction.
Small elevations of body core temperature reliably induce seizures in DS and F/+
). To investigate cardiac function defects associated with generalized tonic-clonic seizures, 1 seizure was thermally induced daily in P24–P30 DS mice and P22–P27 F/+
mice for up to 4 days, and simultaneous video-EEG-ECG records were obtained. Twenty-three DS mice and 9 F/+
mice were tested, and 82 thermally induced seizures were recorded. Four out of 23 (17.4%) DS mice and 4 out of 9 (44.5%) F/+
mice died during these experiments. We first analyzed the combined video-EEG-ECG records of the mice that survived the experiments. The initial seizure discharges observed during elevation of body core temperature were myoclonic seizures, as we reported previously (20
). Only a small portion (2 out of 23) of these mice displayed short ECG pauses (likely to be AV blocks) associated with myoclonic seizures. Further increase in temperature induced generalized tonic-clonic seizures (Figure A). The EEG-ECG records revealed that DS mice experienced a period of bradycardia at the onset of the generalized tonic-clonic seizures, followed by tachycardia, and a second period of bradycardia at the end of the seizures (Figure , A and B). Video records showed that the episodes of bradycardia began at the onsets of the tonic phases of the generalized tonic-clonic seizures (Figure A). Despite the striking bradycardia, the QRS duration and R-wave amplitude were not significantly different from control values during bradycardia or tachycardia in the 19 DS mice that did not die (Figure C). The marked changes in heart rate point to excessive parasympathetic tone at the onset and end of tonic-clonic seizures and increased sympathetic tone during the tonic-clonic seizures as the major contributing factors to changes in cardiac function. The episodes of bradycardia were correlated with the tonic phase of the seizures.
Thermally induced seizures and bradycardia.
Arrhythmia in DS mice that died during thermally induced seizures.
As indicated above, a portion of DS mice (4/23) and F/+:Dlx-Cre+ mice (4/9) died during thermal seizure induction following a generalized tonic-clonic seizure. Each of these mice survived at least 1 thermal seizure induction before death. To examine whether seizure severity predicted susceptibility to death, the duration and Racine score of the fatal and nonfatal seizures were compared in each animal. In DS mice, both fatal and nonfatal seizures were of Racine 5 severity and had similar durations (Figure , A and B). In F/+:Dlx-Cre+ mice, the fatal seizures were more severe than the nonfatal ones (Racine 5.0 ± 0 vs. 3.5 ± 1.0, n = 4; P = 0.001), but the duration periods of the 2 types of seizures were not different (Figure B).
Ictal bradycardia and premature death.
To investigate the functional defects that predict death, the EEG and ECG records of DS and F/+:Dlx-Cre+ mice that succumbed during these experiments were evaluated. The time of death in these records was defined as the point of cessation of cerebral electrical activity, when the total power of the EEG dropped to zero. This also corresponded to the time of cessation of all movements, including respiration. The duration of episodes of ictal bradycardia was longer in mice that died compared with those that survived for both DS mice and F/+:Dlx-Cre+ mice (Figure B). To examine whether ictal bradycardia preceding death was accompanied by impaired cardiac conductance, changes in the indices of cardiac conduction during seizure and during resting behavior 30 minutes before seizure induction were examined. During nonfatal bradycardia in DS mice, the QRS duration increased without change in R-wave amplitude (Figure C). In F/+:Dlx-Cre+ mice, no bradycardia was observed in nonfatal seizures (Figure B). For fatal bradycardia in DS mice, the QRS duration was significantly increased and the R-wave amplitude was significantly reduced (Figure D), whereas in F/+:Dlx-Cre+ mice, the QRS duration was unchanged but the R-wave amplitude was significantly reduced (Figure D).
Reduced incidence of SUDEP after treatment with atropine and N-methyl scopolamine.
To examine whether the periodic ictal bradycardias in DS mice are due to activation of parasympathetic input to the heart, 5 DS mice implanted with EEG and ECG electrodes were tested on 3 consecutive days with an acute i.p. injection of saline on the first day, atropine (1 mg/kg) on the second day, and saline on the third day. On each day, 30 minutes after injection, mice were subjected to the thermal seizure induction protocol, and concurrent video-EEG-ECG recordings were carried out. All of the mice had ictal bradycardia during saline treatment, but treatment with atropine prevented bradycardia in all of the mice (Figure A). This effect was reversible after wash-out of the drug. Similar experiments showed that treatment with propranolol (1 mg/kg, i.p.) did not eliminate the bradycardia in all 5 DS mice tested (Figure B), but combined treatment of atropine and propranolol eliminated bradycardia in all DS mice treated (Figure C). These findings indicate that ictal bradycardia is caused by hyperactivation of parasympathetic input to the heart.
Effects of atropine and N-methylscopolamine on bradycardia and death.
To examine the cause-and-effect relationship between ictal bradycardia and SUDEP, 15 DS mice were chronically treated with atropine via an osmotic pump (10 mg/kg/day) during the age period of their highest incidence of SUDEP (P21–P28), and 20 DS mice were similarly treated with saline for control. Atropine treatment greatly reduced SUDEP compared with controls (Figure E). This finding illustrates the efficacy of chronic treatment with atropine in preventing SUDEP in DS mice. We also tested the effect of acute treatment with atropine in F/+:Dlx-Cre+ mice, which always die following thermally induced Racine 5 generalized seizures. Acute atropine treatment allowed all 5 F/+:Dlx-Cre+ mice studied to survive Racine 5 generalized seizures. Together, these results indicate that prevention of hyperstimulation of the heart by the parasympathetic nervous system is sufficient to prevent ictal bradycardia and SUDEP in these mouse models of DS.
Atropine exerts both central and peripheral anti-muscarinic actions. To ensure that the reduced incidence of SUDEP in DS mice treated with atropine is due to cardiac and not central action of the drug, mice were treated with N-methyl scopolamine, a muscarinic receptor antagonist that does not cross the blood-brain barrier, in both acute and chronic experiments as above. Acute administration of N-methyl scopolamine (1 mg/kg, i.p.) eliminated the bradycardia associated with thermally induced seizures in all 10 treated DS mice (Figure D). Chronic treatment with the drug (10 mg/kg/day, osmotic pump), in the fourth week of life substantially reduced the incidence of deaths in DS mice compared with control saline treatment (Figure E). These findings indicate that peripheral blockade of muscarinic receptors is sufficient to reduce SUDEP in DS mice.