Abstracts

Rationale: Dravet syndrome (DS) is an infantile-onset refractory epilepsy with a high incidence of sudden unexpected death in epilepsy (SUDEP). After non-fatal seizures, DS patients often show severe respiratory and cardiac abnormalities such as decreased O2 saturation, apnea, ataxic and paradoxical breathing, and bradycardia. There is now increasing evidence from both DS mouse models and DS patients that the primary mechanism of death is often seizure-induced respiratory arrest. In adult patients with other types of epilepsy, seizure spread into amygdala coincides with onset of central apnea with concomitant O2 desaturation. Central apnea can also be elicited in epilepsy patients upon stimulation of the amygdala or hippocampus using depth electrodes. Based on these findings, we hypothesized that the amygdala is a critical node in the anatomical pathway by which seizures in the forebrain influence the brainstem respiratory network. Here we examined the role of the amygdala in ictal/postictal respiratory arrest and its possible involvement in SUDEP in a mouse model of DS (Scn1aR104X/+). Methods: Baseline breathing, the hypercapnic ventilatory response (HCVR), and the hypoxic ventilatory response (HVR) were assessed using whole-body plethysmography in DS mice before and after lesioning the central amygdala (CeA). Under surgical anesthesia with isoflurane (1-2%), electrolytic lesions were induced in the CeA of DS mice, after which they were instrumented with a headmount with EEG, ECG and EMG electrodes. After five days of recovery, DS mice were connected to a preamplifier and placed in a plethysmography chamber to noninvasively measure breathing and monitor EEG, ECG, and EMG. Seizures were induced by using a heat lamp by slowly raising body temperature at a rate of 0.5°C every 2 minutes until a seizure occurred with full hindlimb extension and ventilatory arrest or 42.5°C was reached. In a separate set of experiments, naïve (unlesioned) DS mice were placed in a head-out plethysmograph to measure breathing, lightly anesthetized with isoflurane (0.5 - 1%) to minimize movement that could interfere with data collection. Using a stereotactic manipulator, a monopolar electrode was inserted into the CeA to inject electrical current while measuring breathing.The locations of lesions and placement of electrodes were verified post-hoc histologically. Results: CeA lesions did not affect baseline breathing, HCVR or HVR, or prevent heat-induced seizures in DS mice. Preliminary data showed that respiratory arrest and death occurred in control (sham-lesioned) mice following heat-induced seizures in 58.8% (10/17) of cases. In contrast, only 9.1% (1/11) of mice with CeA lesions showed ventilatory arrest and death (p=0.0161). In some regions of the CeA, stimulation caused apnea that was dependent on the frequency and amplitude of stimulation, without causing seizures. In other regions of the CeA, stimulation resulted in an increase of the respiratory rate. At 50 Hz and 500 micro-amps, apnea was induced for as long as 4 minutes without causing death. There was also no reversal of anesthetic immobility, suggesting lack of an arousal response. Conclusions: Our data suggest that different regions of the CeA can modulate breathing in different ways, but it is not directly involved in baseline respiratory rhythm generation or modulation of breathing by chemoreceptors. The CeA is a critical component of the neural pathway required for seizures in the forebrain to influence brainstem function to cause ventilatory arrest and SUDEP. Funding: NIH/NINDS U01-NS0904143 SUDEP Research Alliance