Abstracts

Rationale: Acquired epilepsies are characterized by spontaneous, recurrent seizures that emerge following injury. Brain injuries account for 20-60% of all epilepsy and one third of patients with post-traumatic epilepsy are refractory to current treatment options.  In order to guide the development of new treatment options, we need to know more about the process of seizure initiation, or “ictogenesis”, in chronically epileptic networks. In this study, we investigated the patterns of preictal activity in the hippocampal organotypic slice culture model of post-traumatic epilepsy. Methods: Hippocampal slice cultures become spontaneously epileptic following the widespread axotomy that occurs during slicing. Slice cultures were prepared on P6-8, and were immediately placed on membrane inserts in a glass-bottomed 6-well plate. Organotypic slice cultures were then transferred to the Incuscope: a CO₂ incubator customized to include optics for inverted fluorescence microscopy, a motorized stage for positioning the samples, and a computer-controlled multi-channel perfusion pump, which allowed for the longitudinal study of network activity, with single cell resolution. Calcium dynamics were serially imaged in both principal cells and interneurons using the red calcium sensitive transgenic fluorophore jRGECO, over the course of several weeks. Results: Ictal activity began to emerge over the first week post-injury and by DIV10, we could reliable detect hundreds of interictal spikes and ~10 seizures per hour. We found that each slice culture developed a stereotyped region which drove both spikes and seizures. However, there was considerable variability in the activation pattern of individual neurons within this region. Future studies will aim to parse the contribution of principal cell and interneurons in the preictal buildup to seizure initiation.    Conclusions: The newly developed Incuscope, in combination with genetically encoded calcium indicators, allowed us to follow the activity of individual neurons over the courses of several weeks and to image hundreds of spontaneous seizures. This approach has laid the groundwork for dissecting ictogenesis with single cell resolution.  Funding: NIH