Optimizing Organotypic Slices to Study Epileptogenesis

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DESCRIPTION (provided by applicant): The NINDS has issued U- and R-series RFAs this year to discover drugs to prevent the development of epilepsy after brain injury. The rate-limiting factors in the response to the RFAs are the lack of highly scalable models of epilepsy after brain injury, and the means to quantify epileptogenesis. The hippocampal slice is a compelling model of traumatic brain injury, and organotypic cultures of these slices provide a unique window into recovery after severe shear injury. We re-discovered the utility of the organotypic slice culture as an accelerated model of post traumatic epileptogenesis 20 years after epileptic activity in this preparation was first described. The epilepsy research community lost 20 years of use of this exceptionally powerful model largely because inadequate standardization created a perception of inadequate reproducibility. We propose to optimize the organotypic slice preparation for the study of epileptogenesis, focusing on 3 key questions to maximize its utility: First, what are the best means to quantify epileptogenesis in this preparation? We have developed methods for continuous electrographic recordings and automated seizure detection algorithms that make feasible the parallel processing of arrays of epileptogenic slice cultures. We will determine whether the methods that we have devised for quantification of in vivo epileptogenesis can be applied to these culture arrays. Second, what are the optimum culture conditions for epileptogenesis in slice cultures? This preparation was never optimized for the study of epilepsy, so we need to ascertain that metabolic substrates and products do not limit the quantity of epileptic activity. Third, what surrogate markers can be used to accelerate the use of this system for screening? Although quantification of epileptogenesis from electrographic recordings is the gold standard, our preliminary data suggest that there are potentially promising markers such as extracellular lactate levels and intracellular chloride and calcium levels that can be used as the basis of fluorescence assays that will substantially accelerate throughput with this model. Together, these studies will enable a new, rapid, highly scalable, readily transferrable, and quantifiable assay of post traumatic epileptogenesis that will accelerate both fundamental research into pathogenesis and the search for therapeutic strategies.