Project Summary/Abstract Genetic studies in humans revealed that gain-of-function variants of SCN2A are associated with epilepsy, whereas loss-of-function variants of SCN2A are associated with autism spectrum disorder (ASD). However, about a third of ASD children carrying SCN2A loss-of-function or nonsense variants (collectively referred to as SCN2A deficiency) develop intractable seizures, major comorbidity associated with ASD. How SCN2A deficiency contributes to seizure is largely unknown. To address this problem, we generated a Scn2a-deficient mouse model by gene-trap knockout (gtKO) strategy. Homozygous gene-trap knockout mice have only a quarter of the Scn2a expression level of WT mice, which model severe Scn2a deficiency. Our preliminary patch-clamp recordings reveal that neurons with severe Scn2a deficiency displayed neuronal hyperexcitability. This finding is unexpected, but could open the door for the understanding of the puzzling seizure comorbidity in SCN2A deficiency-related ASD. However, critical gaps exist regarding the possible mechanisms underlying Scn2a deficiency-related neuronal hyperexcitability, its consequences on neural network and seizures susceptibility, as well as the reversibility of seizures-related phenotypes. Filling these gaps would greatly enhance understanding of the mechanisms underlying seizure comorbidity in ASD; and shed new light on the development of effective therapeutic interventions. To this end, here we propose to test an overarching hypothesis that a substantial reduction of Scn2a expression results in increased neuronal excitability related to K channel downregulation, hypersynchronization of in vivo firing, and elevated seizure susceptibility that can be reversed by targeted genetic interventions. We will test our hypothesis at the cellular, circuit, and in vivo levels. In Aim 1, we will assess ex vivo neuronal excitability of Scn2a-deficient mice. In Aim 2, we will determine in vivo neuronal firings and seizure susceptibility of the Scn2a-deficient mice. In Aim 3, we will test targeted genetic interventions. Our study is significant in the following ways: i) SCN2A deficiency is among the leading monogenetic forms of ASD and seizure comorbidity occurs in about 30% of affected ASD patients; ii) The finding that severe SCN2A deficiency resulting in hyperexcitability is potentially paradigm-shifting, and the study of which could reveal key insights regarding seizure comorbidity associated with ASD; and iii) Targeted genetic interventions to be evaluated have clear translational relevance. Our study has the following innovations: i) the use of novel Scn2a deficient mice that reveal unexpected finding of neuronal hyperexcitability; ii) innovative ways to achieve genetic rescue; and iii) the use of cutting-edge technologies including high-density Neuropixels recordings. The applicant is an early stage investigator (ESI), whose team has extensive expertise in sodium channel physiology, genetics, electrophysi...