Abstracts

Rationale: Epilepsy patients are at high risk for SUDEP, and in many cases this is thought to be due to seizure-induced apnea. The mechanisms involved in apnea remain unknown, but evidence has suggested the amygdala plays a critical role in the pathway by which seizures propagate to the brainstem respiratory network. Here we analyzed the role of the amygdala in seizure-induced apnea and death using both Scn1a^R1407X/+ (Dravet Syndrome; DS) mice and Scn1a^R1407X/+/GAD67-GFP mice.

Methods: In DS mice (n=4), the amygdala was stimulated under light anesthesia by injecting current (500 mAmp, 50 Hz) with monopolar electrodes guided by a stereotaxic manipulator while measuring breathing using head-out plethysmography (Location from Bregma: -0.5 rostral-caudal, -2.75 medial-lateral, -4.6 dorsal-ventral). The locations of the electrode were verified post hoc by electrolytic lesion and histology. A 3D map was generated of the portions of the amygdala where breathing was altered.

A second cohort of DS mice were electrolytically lesioned in the rostral amygdala (n=17) or served as control (n=5). Body temperature was increased with a heat lamp by 0.5°C per minute until a seizure was induced, or a maximum of 42.5°C was reached. Temperature of first seizure and fatal seizure were recorded. Again, locations of the electrode were verified post hoc by electrolytic lesion and histology.

A third cohort of DS mice (n=10) and Scn1a^R1407X/+/GAD67-GFP mice (n=4) had their body temperature increased with a heat lamp by 0.5°C per minute until a mild seizure (modified Racine scale 5) was induced. After 60 minutes, mice were perfused, and brains extracted, after which immunohistochemistry against c-Fos expression was performed. WT mice (n=2) and GAD67-GFP mice (n=2) heated to 42.5°C served as controls. Prior to seizure induction, apnea was induced by electrical stimulation of the amygdala in DS mice (n=4) and fluorescent beads were injected to target the location where apnea was induced. Confocal and fluorescence images were taken for analysis.

Results: In the first cohort apnea was successfully induced in mice for the duration of electrical stimulation.

In the second cohort, the temperature of the first seizure was the same for both lesioned (n=17) and control (n=5) DS mice. By the time body temperature had reached 41°C, over 50% of mice in both groups had reached their initial seizure. Respiratory arrest and death occurred in less than 20% of lesioned mice (n=17). In contrast, over 80% of control mice (n=4) reached a fatal seizure ending in respiratory arrest.

In the third cohort, fluorescence images showed greater c-Fos expression in the amygdala of DS mice (n=10) in which a seizure had been induced as compared to control mice with exposure to heat (n=4).

Conclusions: Our results indicate that the rostral amygdala is highly activated by heat induced seizures, and that the amygdala plays a role in modulating breathing. While the amygdala may not play a significant role in seizure induction, it most likely plays a role in fatal seizures ending in respiratory arrest.

Funding: Please list any funding that was received in support of this abstract.: NIH NINDS (U01 NS090414 & U01 NS090407) to GR.