Project Summary/Abstract Elucidating the basic mechanisms by which a normal brain is transformed into an epileptic brain has been a holy grail of epilepsy research for decades. If the mechanisms of epileptogenesis can be understood, then new treatments and therapies can be designed to target these processes to prevent – and possibly cure – epilepsy. While years of research have revealed a multitude of changes that occur during epileptogenesis, one basic problem has been distinguishing changes that mediate epileptogenesis from changes that are associated with the disease, but play no causal role. This problem is evident for almost all existing models of epilepsy, which produce widespread brain damage and cellular changes, thereby making the proximal cause of the disease difficult to ascertain. For the present proposal, we utilize a novel mouse model of epilepsy generated in the first term of this grant, in which spontaneous cortical seizures develop following conditional, inducible deletion of the mTOR pathway inhibitor phosphatase and tensin homologue (PTEN) from >9% of hippocampal dentate granule cells. The effect of this deletion – to induce the abnormal integration of newborn granule cells – is important because abnormal newborn granule cells are a hallmark pathology of temporal lobe epilepsy, and are suspected of causing the disease. Our model provides direct evidence that abnormal granule cells can cause epilepsy, and creates an opportunity to understand how they cause the disease. Work with this model has led us to hypothesize that spontaneous seizures in temporal lobe epilepsy result from the combinatorial effects of intrinsically hyperexcitable granule cells and impaired inhibitory control of these cells. To test this two-hit model of epileptogenesis, we will make full use of a unique feature of our PTEN knockout model: the ability to manipulate the number or “load” of abnormal granule cells. We will generate animals in which 5-8% of granule cells lack PTEN. These animals exhibit abnormal hippocampal physiology, but not overt cortical seizures. This constitutes the first “hit”. We will then challenge these altered circuits by silencing distinct classes of hippocampal inhibitory interneurons in vitro and in vivo – the second hit. We predict that disinhibition will act synergistically with PTEN knockout granule cells to destabilize the hippocampal circuit and promote seizures. The studies will provide a proof-of-concept test for how temporal lobe epilepsy develops, and will provide insights into therapeutic strategies.