Abstracts

Rationale: Patients with refractory epilepsy are at increased risk of sudden unexpected death in epilepsy (SUDEP). SUDEP is the leading cause of death in these patients, but its exact etiology is unknown. A potential risk marker for SUDEP is post-ictal generalized EEG suppression (PGES), a period of EEG attenuation that follows some seizures. The presence and duration of PGES may signify increased SUDEP risk, but the mechanisms underlying PGES are unknown. PGES is consistently observed after maximal electroshock (MES) and amygdala kindled seizures in mice in our lab. Data from preliminary studies suggest that pharmacologically increasing serotonergic (5-HT) tone reduces PGES duration, but the underlying network remains unknown. We hypothesized that the dorsal raphe nucleus (DRN) may be involved in PGES because the DRN sends 5-HT projections broadly to regions associated with epileptogenesis, sleep/wake regulation, and arousal, and neuronal activity in the DRN is reduced by seizures. Therefore, we further hypothesized that stimulating the DRN prior to a seizure may reduce PGES duration and reduce mortality. Methods: Six C57BL/6J (3 male, 3 female; 8-12 weeks old) and eight Pet1-Cre and phenotypically wild type littermate mice (8 per genotype, 4 male, 4 female; 8-12 weeks old) were implanted with EEG/EMG electrodes and a bipolar stimulating/recording electrode in the right basolateral amygdala (AP: -1.3 mm; ML: -2.8 mm; DV: -4.7 mm). Mice also received either a microdialysis cannula or an injection of a Cre-dependent viral vector of channelrhodopsin2 and an optic optical fiber directed at the DRN (AP: -4.6 mm; ML: 0 mm; DV: -3 mm). After recovery, animals underwent amygdala afterdischarge threshold determination, and were kindled with the threshold current (1 sec train of 1 msec square wave pulses at 60 Hz; 2x/day) until three consecutive Racine 4 seizures were observed. The DRN was stimulated in two ways. Seizures were induced during wakefulness following either focal chemical (45 µl/min, 6.8 pH artificial cerebrospinal fluid,10 min) or optogenetic (473 nm, 4 Hz, 10 mW, 2% duty, 10 s ON / 10 s OFF, 10 min) DRN stimulation. Seizure and PGES duration were assessed. In a separate set of experiments, six (2 male, 4 female; 8-12 weeks old) C57BL/6J mice were implanted with EEG/EMG electrodes and a microdialysis cannula into the DRN. Mice received pretreatment with chemical DRN stimulation as above and then underwent MES induction during wakefulness (0.2 s, 60-Hz sine wave pulses). Mortality, seizure duration, and seizure severity were determined off-line by post hoc video review. After experimentation, all animals were intracardially perfused and implant placements were verified histologically. Results: Focal chemical and optogenetic stimulation of the DRN prior to seizure induction via amygdala stimulation in kindled animals decreased PGES length. Chemical DRN stimulation also reduced mortality in the MES model. There was no difference in seizure severity between animals that survived vs. died. Seizure length was unaffected by either manipulation. Conclusions: These data suggest that the DRN is involved in PGES generation and in seizure-induced mortality. Future experiments will determine the downstream circuitry and 5-HT receptor subtypes underlying the effect of DRN stimulation on PGES generation. Better understanding possible SUDEP risk indicators such as PGES may help in identifying and protecting high risk patients from SUDEP. Funding: This work was supported by the Beth L. Tross Epilepsy Professorship and NIH/NINDS R01 NS095842.