Brain injury is a common cause of medically intractable epilepsy. Because we do not understand the underlying pathophysiology, we can’t reduce the burden of medical intractability. The overarching goal of this research program is to exploit a recently developed large animal gyrencephalic model of neocortical post-traumatic epilepsy (PTE) to elucidate a mechanism of neocortical epileptogenesis: chronic compromise of the ionic basis for GABA-mediated inhibition. Disinhibition is a fundamental element of ictogenesis, but thus far we have not found the mechanism of disinhibition in chronic epilepsy. There are two salient clues: first, anticonvulsants that increase GABA conductance often do not ameliorate PTE; and second, studies in human intractable epilepsy have found a positive, disinhibitory shift in the reversal potential for GABAA receptor-mediated membrane currents (EGABA). Here we will test the hypothesis that glial reconstruction of the brain’s extracellular matrix after injury results in increased displacement of extracellular chloride (Cl-o) by the sulfate moieties of the proteoglycans that comprise the new matrix. To test this hypothesis, we will employ a large-animal PTE model in which local epileptogenesis occurs after well-characterized neocortical injury (Project 1 and Large Animal Core). This localization of epileptogenesis is critical for correlation of changes in Cl-o, network structure, and epilepsy. In this injured neocortical area, we will use longitudinal 2-photon in vivo calcium imaging to test for the network changes expected from disinhibition (Project 2 and Microscopy Core). In the injured neocortical area, we will use 2-photon Fluorescence Lifetime Imaging (FLIM) and newly-synthesized Cl- indicators to test for changes in the local Cl-o and intracellular chloride (Cl-i ) that would drive disinhibition by depolarizing EGABA and reducing the efficacy of shunting inhibition (Project 3). The 3 projects have a common third aim: to correlate epileptogenesis (Project 1) with network disinhibition (Project 2) that are associated with chronic changes in Cl-o and Cl-i (Project 3). Together, these Projects and Cores will test a novel mechanism of epileptogenesis and medical intractability that could be ameliorated by short-term inhibition of the disassembly of the brain’s extracellular matrix at the time of injury using MMP inhibitors whose efficacy will be screened in Project 3, Aim 2.