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

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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

*