Project Summary This proposal investigates the hypothesis that stem cell-derived progenitors of GABAergic inhibitory interneurons will treat epilepsy in a validated mouse model of the devastating epileptic encephalopathy known as Dravet syndrome (DS). Characterized as an infantile-onset epilepsy that leads to severe autistic-like cognitive impairment with high rates of sudden unexplained death (SUDEP), DS is caused by a heterozygous loss-of- function mutation in SCN1A encoding the type 1 voltage-gated sodium channel. Although a mechanistic basis of the syndrome remains poorly understood, studies using animal models strongly suggest that Scn1a mutations selectively impair PV and SST inhibitory interneurons, and conditional deletion of Scn1a in forebrain interneurons was previously shown to fully recapitulate the epileptic phenotype. This selective impairment of interneurons makes DS a promising target for the development of therapy based on transplantation of exogenous interneurons, which can migrate long distances from the injection site and integrate synaptically with surrounding cortical interneurons (IN’s) within host brain. This unique ability has driven the successful use of interneurons derived from fetal tissue to treat acquired epilepsy in rodent models. However, it remains unclear whether a rodent model of a genetic epilepsy can be treated effectively with interneurons that are derived from stem cells, a more clinically applicable viable alternative to fetal cells. Additionally, while the physiological roles of endogenous PV and SST cells are relatively well established, it remains unknown how engrafted PV- and SST- fated precursors mature and influence host activity. To address these important questions, we will study the effect of transplants at the level of single cells, circuits, and whole animals. We hypothesize that mouse embryonic stem cell (mESC)-derived interneuron transplants will synaptically integrate into the epileptic brain of DS mice, improve circuit-level disinhibition in vitro, and suppress seizures in vivo. In aim 1, I will use whole-cell patch-clamp electrophysiology in acute brain slices to determine whether transplanted PV and SST interneuron transplants demonstrate intrinsic maturity and influence host activity at the level of interneuron subtype-specific microcircuits. In aim 2, I will assess the inhibitory influence of transplants in vivo by first using optogenetics and calcium imaging to assess inhibition of endogenous excitatory neurons, and then assessing the transplants’ effect on seizures in DS mice.