PROJECT ABSTRACT Up to 30% of individuals with epilepsy have intractable seizures. It is unclear how mechanisms underlying seizure onset and propagation operate regionally in intractable focal epilepsy, in which tissue involvement is heterogeneous and regions play different roles in seizure initiation, propagation and resistance to spread. Better understanding is needed of the biology underlying the seizure focus versus the penumbra, where excitatory discharges appear on EEG but neuronal response is limited by intact inhibition. Imbalance of inhibitory and excitatory neuronal inputs (I/E imbalance) is a primary factor underlying seizure generation and propagation, particularly interneuron dysfunction and interneuron/pyramidal cell interaction. I/E imbalance influences seizure generation in a wide array of epilepsy pathologies; investigation of I/E imbalance and interneuron function can thereby reveal pathways common to many types and causes of epilepsy. We hypothesize that cell-type specific alterations result in I/E imbalance in brain resected during surgery for intractable focal epilepsy, and that these transcriptional changes will correlate with histopathology and regional EEG. We will test this by performing single nucleus RNA sequencing (snRNAseq) on MRI-guided electrophysiologically-localized biopsies from patients undergoing epilepsy surgery. This will provide site-specific correlation of cell type-specific transcriptomic profiles with histological, radiographic, and electrophysiologic alterations. We will sample the seizure focus and penumbra to determine the differences in cellular composition and cell type-specific expression profiles, and identify alterations involved in seizure generation and propagation. Although we will focus on interneuron profiling and I/E imbalance, snRNAseq will provide data on the diversity of cell types. In AIM 1 we will use snRNAseq to identify differential expression of genes in seizure focus vs. penumbra in human brain resected during surgery for intractable focal epilepsy, focusing on interneuron vs excitatory pyramidal cell transcriptional alterations. Microelectrode recordings will provide the spatial trajectory of a seizure and define focus and penumbra. We will identify region- and cell type-specific alterations by comparing transcriptional profiles across EEG-characterized samples. We will thereby identify molecules and pathways differentially expressed in seizure focus vs. penumbra, highlighting processes likely to be primary to seizure generation. In AIM 2 we will perform immunohistochemical analyses of top target molecules identified in Aim 1, as well as molecules previously identified to play a role in I/E imbalance in epilepsy and seizure generation. Our studies will thereby provide microscopic resolution of cellular and subcellular patterns of expression within MRI-guided electrophysiologically localized biopsies. Illuminating the regional pathophysiology of seizures in intractable focal epileps...