Abstracts

Rationale: Mutation of the conserved arginine residue, R1648H, in the voltage sensor of domain four (S4/D4) of SCN1A sodium channels causes Genetic Epilepsy with Febrile Seizures plus (GEFS+) in humans. We have previously shown that the mutation reduces activity of Scn1a (Na_v1.1) channels in cortical interneurons using R1648H knock-in mice. This led to the hypothesis that reduced interneuron function alters cortical circuitry that eventually leads to spontaneous seizures and premature death of homozygous knock-in mice. Our aim was to study the dynamics of network activity at high resolution in order to bridge the gap between our single cell data obtained previously and the seizure phenotype of the mutant mice. Methods: We used a functional mapping technique combining laser scanning photostimulation and voltage-sensitive dye (VSD) imaging to study the activities of neuronal populations in the GEFS+ mouse model. We isolated coronal slices from the visual cortex (V1) from P15 mutant and wild-type mice and stained with voltage sensitive dye. The recording solution contained caged glutamate that was uncaged by 355 nm laser in order to activate a discreet population of neurons. The signal from the dye was used as a measure of spatio-temporal spread of evoked excitability across layers. The initial phase of activation is defined as the first 30 ms after glutamate uncaging and peak phase is next 30 ms when the signal reaches its peak. The total laminar output after stimulation of each layer in wild-type and mutant mice was quantified by total number of activated pixels in initial and peak phase.Results: We found that there was almost 50% greater evoked output across layers in heterozygous and homozygous mice compared to wild-type upon stimulation at layers 2/3 and layer 4 but not upon stimulation of layer 6 that only has local projections. We also noticed that the R1648H homozygous mice demonstrated slower rise in activity initially after stimulation compared to heterozygous, but by peak phase evoked a greater burst of synchronous excitatory output across layers. Our results indicate that there is increased cortical excitability in GEFS+ mutants, probably due to reduced inhibitory control. Similar results were observed when mapping excitatory input connections of layer 4 pyramidal cells using single cell patch clamp combined with laser scanning photostimulation.Conclusions: In this study we demonstrate that the GEFS+ SCN1A mutation leads to increased excitability at a neuronal population level as predicted by the single cell studies performed previously. We speculate that there is an activity threshold, below which we only observe increased susceptibility to induced seizures as seen in the heterozygous mice. In the homozygous mice, the excitability threshold is probably exceeded leading to spontaneous seizures and premature death.