PROJECT SUMMARY SCN8A epileptic encephalopathy (EE) is a severe epilepsy syndrome resulting from de novo gain-of-function mutations in the SCN8A gene, which encodes the voltage-gated sodium channel Nav1.6. Nav1.6 is expressed in both excitatory and inhibitory neurons and plays a critical role in action potential (AP) generation and propagation in both. Multiple previous studies have indicated that a gain-of-function Nav1.6 mutant results in hyperexcitability of excitatory cells. However, significantly less is known about how mutant Nav1.6 may impact inhibitory interneurons. Proper function of inhibitory interneurons is essential to constrain activity of excitatory neurons, and their dysfunction has been linked to various genetic epilepsy syndromes. The most numerous inhibitory interneuron subtypes are parvalbumin-positive (PV) and somatostatin-positive (SST) interneurons. Previous work in our lab has shown deficits in SST interneurons in models of SCN8A EE. PV interneurons play a crucial role in Dravet Syndrome, a genetic epilepsy syndrome caused by mutations in the sodium channel Nav1.1, and they may play a role in temporal lobe epilepsy. Despite this, there have been no studies in the SCN8A EE field examining the effect of mutant Nav1.6 expression on PV interneuron physiology and the role that they may play in seizures. Using global (Scn8aD/+) and conditional (Scn8aW/+-PV; expression solely in PV interneurons) mouse models of SCN8A EE, this proposal seeks to test whether PV interneuron excitability is affected by mutant Nav1.6, and if this impacts overall network excitability and seizure susceptibility. My preliminary data suggest that PV interneurons possessing mutant Nav1.6 have increased persistent sodium currents and reduced excitability at high-firing frequencies via a state of action potential failure known as depolarization block. I have also observed that conditional expression of a patient derived SCN8A mutation solely in PV interneurons leads to spontaneous seizures in mice, further indicating the importance of this interneuron subtype within SCN8A EE. In aim 1 of this proposal, I will record WT, Scn8aD/+, and Scn8aW/+ PV interneurons to characterize any alterations in the intrinsic excitability, voltage-gated sodium currents, and synaptic physiology of PV interneurons. This will clarify the impact of gain-of-function SCN8A mutations on PV interneuron function and network excitability. In aim 2, using a Cre-dependent shRNA, I will test whether genetic knockdown of Nav1.6 specifically in PV interneurons will rescue their aberrant excitability and if this will impact seizure frequency in Scn8aD/+ mice. Overall, completion of these aims will allow for a comprehensive characterization of PV interneurons in SCN8A EE and will assess whether the specific targeting of these interneurons might provide a novel therapeutic target.