Abstracts

Rationale: Sudden unexpected death in epilepsy (SUDEP) is the most frequent cause of death in epilepsy patients, Dravet Syndrome (DS) patients being particularly affected by this premature death. Recently, peri-ictal respiratory dysfunction has been witnessed in DS patients, which may contribute to risk of SUDEP. Prior work in mouse models suggests that DS may develop due to preferential dysfunction of interneurons, particularly parvalbumin (PV) neurons, with decreased synaptic inhibition and hyperexcitability. Outside of cortex and hippocampus little investigation has been performed on potential network changes. Investigation into respiratory-related subcortical areas may be necessary to determine a mechanism for SUDEP in this model. The extended amygdala, composed of the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), is a relay connecting higher cortical and limbic areas to brainstem respiratory nuclei. Manipulations of the extended amygdala, in both human and animal models demonstrates powerful effects on autonomic and respiratory function. I hypothesize that with repeated seizures in DS there is increased excitability in the afferent pathway from the extended amygdala neurons to the brainstem, which puts DS patients acutely at risk for SUDEP.  Methods: Experimental heterozygous F1 mice generated by breeding 129.Scn1a+/− mice with wild-type C57BL/6J mice were used along with their wildtype F1 littermates. Using whole cell patch clamp electrophysiology, recordings were performed from P18-25 mice from ex vivo BNST coronal slices to measure both evoked and spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) as well as measurements of short term plasticity (paired pulse facilitation) and postsynaptic receptor components (AMPA and NMDA receptor ratios). Additionally, current clamp recordings were performed evaluating changes in intrinsic excitability including resting membrane potential, input resistance, action potential (AP) threshold and AP kinetics. Finally, immunohistochemistry was performed to evaluate neuronal c-fos activation in the BNST one hour after a seizure in and to quantitate scn1a expression in the BNST in DS animals and wildtype littermates. Results: We found that there is enhanced excitability in BNST neurons in unbiased recordings of these neurons in the DS model compared to wildtype littermates. Specifically, there was a significant decrease in both frequency of sIPSCs (1.08 +/- 0.22Hz DS n=9, 5.49 +/- 2.87 Hz WT n=14) and amplitude (10.4 +/- 0.97 pA DS n=9, 14.3+/0 0.83 pA n=14 WT, p<0.01) without a change in sEPSC frequency or amplitude, suggesting a relative disinhibited state. This was combined with an enhanced AMPA/NMDA ratio in DS animals (3.3 +/- 0.4 DS n=14, 1.9 +/- 0.8 WT n=7). Preliminary data also suggest an increased frequency in firing of action potentials in DS animals (35.5 +/- 11.7 DS n=3, 16.8 +/-3.4 WT n=5). Taken together, this suggests that there is a loss of inhibition in the BNST neurons in DS animals, combined with increased excitability. We feel that this is due to extrinsic effects of the scn1a mutation, as our immunohistochemistry results show little scn1a in the BNST, which is expected given the lack of PV neurons in the region. However, preliminary c-fos studies shows robust BNST neuronal expression after both spontaneous and hyperthermic seizures. Frequent activation of the BNST through seizures could drive homeostatic plasticity (AMPA/NMDA ratio increase) and alter excitability. Conclusions: The finding of increased excitability and evidence of plasticity/new synapse formation) in BNST neurons in DS animals suggests enhanced output from the BNST neurons. Given the output of these neurons to brainstem respiratory regions, we feel that loss of BNST bidirectional control of brainstem activity in DS may promote SUDEP, with more work yet to be done to determine changes in specific output projections. Funding: Taking Flight Award, Citizens United for Research in Epilepsy; Center for SUDEP Research (CSR) Pilot Grant NINDS; AES Junior Investigator Research Award