1) Deletion of the SCN1A gene in GABAergic interneurons mediated by the Dlx5,6Cre locus leads to increased susceptibility to seizures induced by pentylenetetrazol (PTZ) in mice, including shorter latency to seizures, longer duration of seizures, and increased fatality. 2) Preliminary analysis found an association between increased attenuation of EEG waves after seizures and sudden unexpected death in epilepsy (SUDEP) in a Dravet syndrome mouse model, suggesting EEG attenuation could be a predictive biomarker for SUDEP risk. 3) The study aims to further compare the Dravet syndrome mouse model to a focal cortical dysplasia model to examine shared physiological biomarkers and mechanisms of

1. Deletion of Scn1a within the
forebrain only (Dlx1,2 locus) is
sufficient to exhibit the DS
phenotype (Cheah et al., 2012)
Can a wider selective deletion
beyond the forebrain
exacerbate the DS phenotype?
Create a mouse model with a
conditional deletion in the
forebrain, brain stem, and
cerebellum (Dlx5,6 locus)
Background Methods
Aims
Results: PTZ Susceptibility
Results: EEG Attenuation
Future Directions
Conclusions
Susceptibility to PTZ-Induced Seizures and Postictal
EEG Attenuation in Dravet Syndrome
Elizabeth Krawczak1, Sarah Hanna, Franck Kalume2, PhD
1Pitzer College, Claremont, CA; 2Seattle Childrens Research Institiute, Seattle, WA
Dravet Syndrome (DS)
• An infantile-onset intractable epilepsy
• Associated with heterozygous loss-of-function
mutations in SCN1A (Catterall et al., 2010)
• SCN1A is a gene encoding for type 1 voltage-gated
sodium (NaV1.1) channel (Catterall et al., 2000)
Conditional Deletions of Scn1a: DLX Genes
• DLX genes (dlx1, dlx 2, dlx 5, dlx6) act as key regulators of
GABAergic interneuron migration, differentiation, and survival
(Long et al., 2009).
Aim #1: PTZ Susceptibility
Examine the susceptibility to pentylenetetrazol
(PTZ)- induced seizures in mice carrying Dlx5,6Cre
mediated knock out of Scn1a.
Aim #2: EEG Attenuation and SUDEP
Identify the role of postictal cerebral shutdown as a
biomarker for sudden unexpected death in epilepsy
in DS mice
Experimental design
SUDEP
Respiratory
compromise
Cerebral
shutdown
Cardiac
dysfunction
PTZ Susceptibility:
• Pentylenetetrazol (PTZ) is an antagonist
of the GABAA receptor, used to induce
seizures
• Compared Dlx5,6Cre+ and control mice at
convulsive (60 mg/kg) and sub-convulsive
(20 mg/kg) doses
• 30 min video recording and behavioral
analysis using modified Racine Scale (A.
Luttjohann et al., 2009)
EEG Attenuation and SUDEP:
• Subdural EEG recording
performed after convulsive PTZ
injection
• Compared postictal EEG wave
power in fatal and non-fatal
seizures
SUDEP
• Sudden Unexpected Death in
Epilepsy
• DS has high incidence of SUDEP
proceeding generalized tonic
colonic seizure (GTCS)
• Postictal EEG attenuation
observed in the context of
SUDEP
The SUDEP Triangle:
Figure 1. Example postictal EEG-ECG (black-blue) traces and EEG
power illustrating a seizure, postictal EEG attenuation, bradycardia,
and death (Kalume et al., 2013)
Convulsive Dose:
- Incidence (% mice to have GTC)
- Latency (time to first GTC)
- Duration (% time alive spent in GTC)
- Fatality (% died during experimentation)
Sub-Convulsive Dose:
Figure 4. Decreased latency of PTZ-seizures in Dlx5,6Cre mice. Mean (± SEM) latency
(sec) to first sustained GTC seizure where (Left) Dlx5,6Cre+ mice (n = 10, M = 513 ± 258)
experience a significant decrease in latency to seizure onset compared to control mice (n = 13,
M = 795 ± 317). t-test, *p = .033. (Right) Susceptibility plot of percent mice without a GTC
seizure over time.
Figure 2. Higher fatality in Dlx5,6Cre mice exposed to PTZ. Percent fatality
during PTZ experimentation (Left) illustrating higher mortality in Dlx5,6Cre+ mice
(n = 13) than control mice (n = 18) mice. Chi-square, *p = .026. (Right) Survival
plot of percent mice alive.
Figure 3. Dlx5,6Cre mice experience longer
GTC seizure when exposed to convulsive
dose of PTZ. Mean (± SEM) percent of time
spent in PTZ-induced GTC seizure illustrating
that Dlx5,6Cre+ mice (n = 10, M = 12.7 ± 13.1)
experience longer GTC seizures than control
mice (n = 13, M = 5.1 ± 6.0 percent). t-test, *p
= .037.
Summary
Deletion of SCN1A in GABAergic interneurons
mediated Dlx5,6Cre locus in convulsive dose
leads to significant:
1) Decrease in latency of PTZ-induced seizures
2) Increase in duration of PTZ- induced seizures
3) Increase in fatality
But, did not lead to significant changes in seizure
incidence
In sub-convulsive dose:
1. GTC seizures
2. Increase in incidence of myoclonic activity
PTZ Susceptibility:
• Dlx5,6Cre+ mice show increased susceptibility to PTZ-
induced seizures compared with controls, possibly due
to a decreased seizure termination mechanism
• Future experiments will compare Dlx5,6 data to Dlx1,2
and het Scn1a KO mice to characterize severity of DS
phenotype
EEG Attenuation and SUDEP:
• Preliminary analysis suggests an association between
increased postictal EEG attenuation and SUDEP
• Suggests EEG attenuation could be used as a
predictive clinical biomarker of SUDEP
Aims coming together: DS model to be compared with
focal cortical dysplasia (FCD) model
• Studying seizure susceptibility and SUDEP in DS
mice with PTZ allows us to compare across different
mouse genotypes
• FCD is a malformation of cortical development
leading to intractable epilepsy
• Do the two mouse genotypes exhibit similar
physiological biomarkers and mechanism of SUDEP?
Acknowledgements
I would like to thank Dr. Franck Kalume for his guidance and support, as well
as Sarah Hanna for her research assistance. I would also like to thank Dr.
Thomas Borowski and the Claremont Colleges Neuroscience Summer
Research Fellowship for this opportunity.
1. Catterall, W. A., Kalume, F., & Oakley, J. C. (2010). NaV1. 1 channels and epilepsy. The Journal of physiology, 588(11), 1849-1859.
2. Cheah, C. S., Frank, H. Y., Westenbroek, R. E., Kalume, F. K., Oakley, J. C., Potter, G. B., ... & Catterall, W. A. (2012). Specific deletion of NaV1. 1 sodium channels in inhibitory interneurons causes seizures and
premature death in a mouse model of Dravet syndrome. Proceedings of the National Academy of Sciences, 109(36), 14646-14651.
3. Kalume, F., Westenbroek, R. E., Cheah, C. S., Yu, F. H., Oakley, J. C., Scheuer, T., & Catterall, W. A. (2013). Sudden unexpected death in a mouse model of Dravet syndrome. The Journal of Clinical
Investigation, 123(4), 1798–1808. doi:10.1172/JCI66220
4. Long, J. E., Swan, C., Liang, W. S., Cobos, I., Potter, G. B. and Rubenstein, J. L.R. (2009), Dlx1&2 and Mash1 transcription factors control striatal patterning and differentiation through parallel and overlapping
pathways. J. Comp. Neurol., 512: 556–572. doi: 10.1002/cne.21854
5. Lüttjohann, A., Fabene, P. F., & van Luijtelaar, G. (2009). A revised Racine's scale for PTZ-induced seizures in rats. Physiology & behavior, 98(5), 579-586.
Figure 6. Marked reduction of post-ictal EEG
power in DlxCre+ mouse. Postictal power density
as a proportion of baseline activity illustrating a
decrease in postictal power density in fatal seizures
as compared to non-fatal seizures in a Dlx5,6Cre+
mouse (n = 1)
• Power density defined as total
EEG wave power divided by the
EEG section duration in seconds
• In one Dlx5,6Cre+ mouse,
postictal power density is smaller
in a fatal seizure compared with a
non-fatal seizure
*
0
10
20
30
40
50
60
70
Dlx5,6Cre+ Control
PercentExperiencingGTC
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22 2324 25 26 27 28 29 30
AverageNumberofRacine3s
and4s
Time (min)
Control
DlxCre+
Figure 5. (Top) Incidence of PTZ-induced GTC seizure at sub-convulsive
doses, showing that Dlx5,6Cre+ mice (n = 5) experience PTZ-induced
GTC seizures while control mice (n = 5) do not. Chi-square, *p < 0.05.
(Bottom) Dlx5,6Cre+ mice show an increase in mean myoclonic activity
over time.
Table 1: Seizure Intensity Stages for PTZ scale
*