Abstracts

Rationale: The novel anti-seizure medication fenfluramine (FFA) is interesting for its effect on sudden unexpected death in epilepsy (SUDEP), as it is effective in decreasing seizure-induced mortality in patients and different mouse models - both Dravet Syndrome models and the audiogenic seizure mice (AGS) DBA1. While the mechanism is unknown, rescue of seizure-induced respiratory dysfunction is a possible mechanism. Our goal is to explore this hypothesis and further evaluate circuit level effects of FFA on respiratory-related brain regions in the AGS mice.

Methods: We utilize whole body plethysmography (WBP) to measure respiratory activity pre- and post-ictal and before and after FFA administration. After acclimation to the WBP chamber, baseline respiratory status and hypercapnic ventilatory response (HCVR) in the presence of 7% CO2 gas is measured both pre and post ictally. Seizures are induced in the DBA1 ( >95dB broad tone). We investigated FFA administration at 10mg/kg and 20mg/kg intraperitoneal injection or saline. Seizure severity and latency to are measured. To measure circuit level effects of respiratory regions, DBA1 mice were first primed to have repeated convulsive seizures then an AAV targeting neurons to express GCamp8f is injected stereotaxically into the bed nucleus of the stria terminalis (BNST), a region our group has shown can modify SUDEP in the DBA1 mice. After allowing 3 weeks for expression, ex vivo slices containing the BNST are imaged during bath application of 10uM FFA and analyzed for df/F in neurons. Finally, to further explore the effect of FFA on BNST activity, whole-cell patch clamp experiments were performed measuring spontaneous inhibitory currencts (sIPSC) during bath application of 10uM FFA.

Results: At the higher dose of FFA (20mg/kg), seizures were unable to be induced with audio exposure, consistent with the literature and only 1/5 mice had convulsive seizures with the 10mg/kg dose. Preliminary analysis suggests no changes in baseline respiratory data, analysis of post-ictal data is ongoing. Ex vivo calcium imaging showed increase in calcium transients following bath application of FFA (n=29 neurons from 2 slices from 2 animals, p< 0.01) and whole-cell patch clamp found preliminary evidence for increase in spontaneous inhibitory postsynaptic currents (sIPSCs) following application of 10uM FFA (30 +/-11% increase frequency of events from baseline, n=2 cells).