Dynamic two-photon calcium imaging and optogenetic manipulation of epileptic brain circuits in an experimental model of temporal lobe epilepsy

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PROJECT SUMMARY This mentored career development award proposal describes an integrated training program designed to advance my career towards the goal of running an independent R01-funded biomedical research laboratory focused on the study of epilepsy. Currently, there is no way to prevent epilepsy in at-risk individuals prior to the appearance of seizures, and there are limited treatment options for patients with medically intractable epilepsy. With the guidance of my mentor, Dr. Coulter, I have designed a training plan to successfully learn and apply a coordinated, powerful set of state-of-the-art techniques – including electrophysiology, optogenetics, and two- photon calcium imaging – in vitro and then in awake, behaving experimental animals in vivo. The proposed research tests the hypothesis that brain circuit dysfunction in a well-established model of epilepsy is due to abnormal activity of a defined subtype of inhibitory interneuron, the fast-spiking cells (“FS cells”). This multimodal analysis of circuit-level mechanisms of epilepsy will yield novel results that will contribute to the development and application of novel therapeutic strategies to prevent and treat epilepsy. Candidate: I am currently Assistant Professor in the Division of Neurology at The Children's Hospital of Philadelphia (CHOP) and Departments of Neurology and Neuroscience at The Perelman School of Medicine at the University of Pennsylvania (UPenn). I am an M.D./Ph.D. physician-scientist with a strong background in neuroscience, having received a Ph.D. in Physiology & Neuroscience from NYU in the laboratory of Dr. Bernardo Rudy. I completed a five-year clinical training program in pediatric neurology at CHOP/UPenn and now take care of children with epilepsy in General Neurology and Neurogenetics Clinic at CHOP. This proposal builds on my long-standing interest in the neurobiology of disease and established interests in synaptic inhibition and GABAergic inhibitory interneurons in the cerebral cortex. This K08 award will provide me with critical training and support to insure a successful transition to independence and long-term achievement and productivity as a neuroscientist and academic pediatric neurologist in the field of epilepsy. My goal is to become an R01-funded independent investigator studying epilepsy in mouse models to inform the development of mechanistically oriented therapies that could be translated to, and transform, patient care. Environment: My mentor is Dr. Douglas Coulter, an established investigator in the field of epilepsy and a pioneer in the application of dynamic imaging methods to the study of epilepsy mechanisms. Dr. Coulter is Director of the Center for Dynamic Imaging of Nervous System Function at CHOP/UPenn and the Translational Research Epilepsy Program at CHOP; he has multiple RO1 grants studying epilepsy. Dr. Coulter also has a robust track record of mentoring trainees who have gone on themselves to be leaders in the field of epilepsy. His laboratory is located in the Abramson Research Building, where the 4th and 5th floors are dedicated to neuroscience research and include a collaborative group of highly successful scientists who are interested in and committed to my career development and success. Dr. Coulter and I have constructed an outstanding mentorship team to guide the execution of the proposed studies and my overall career development. I will attain mastery in the clinical field of epilepsy neurogenetics under the guidance of Eric Marsh, M.D., Ph.D., Head of the Section on Neurogenetics, Division of Neurology, at CHOP, who also runs an R01-funded basic neuroscience laboratory. Training will occur at CHOP/UPenn, an academically enriching neuroscience community with extensive resources and opportunities for scientific interaction, including a wide range of available coursework and multiple ongoing neuroscience-, neurology-, and epilepsy-related seminar series. My career development plan involves rigorous training in dynamic imaging, optogenetics, and the study of epilepsy in animal models, coursework in crucial subject areas, as well as formal and informal training in how to properly conduct science and run a research laboratory. This application is supported enthusiastically by the Division of Neurology at CHOP and Department of Neurology at UPenn. Research: My preliminary results show that there is abnormal GABAergic synaptic inhibition in the hippocampus in a well-established animal model of temporal lobe epilepsy; namely, with failure of the so-called “dentate gate.” Rather than being a general failure of inhibition, I have determined that a defined subset of GABAergic inhibition interneuron in dentate gyrus exhibits abnormal activity in epilepsy. This proposal will build on my preliminary data to test the hypotheses that: (1) the mechanistic basis of the dentate gate is feed-forward inhibition specifically provided by fast-spiking interneurons, and (2) manipulation of FS cell activity in the epileptic brain using optogenetics can reconstitute normal circuit activity. I predict that targeted silencing of fast-spiking cells in control conditions will reproduce epileptic circuit pathology and augmenting the activity of these cells in epileptic brain will recover normal inhibition. These outcomes will provide novel information regarding the normal function of fast-spiking interneurons and role of synaptic inhibition in dentate gyrus, as well as establishing important mechanistic contributions to the pathogenesis of temporal lobe epilepsy. This mentored career development award will ultimately position me to translate the insights gleaned from basic neuroscience research to inform and motivate future attempts at the targeted treatment of epilepsy based on manipulation of GABAergic interneurons.