Final Report Summary - HUBS IN EPILEPSY (Functional connectivity and the role of hub neurons in epilepsy) Epilepsy is characterized by recurrent seizures and brief, synchronous bursts called interictal spikes, present in electroencephalogram (EEG) signals in-between seizures. Although the temporal dynamics of epileptiform synchronizations are well described in vivo at the macroscopic level using electrophysiological approaches, less is known about how spatially distributed microcircuits contribute to this activity.The Hubs in Epilepsy project focused on the hypothesis that changes in network structure and/or hub neurons are involved in the development of epileptiform activity. We addressed this hypothesis through two complimentary objectives:1) The detection of differences in neuronal dynamics and functional network structure derived from calcium imaging of epileptic tissue both in vitro and in vivo.2) Monitoring the survival and features of early-born hub neurons in chronically epileptic tissue using histological approaches.In order to study neuronal dynamics and network structure in epileptic tissue, we utilized the pilocarpine mouse model of temporal lobe epilepsy (TLE). This chronic model of epilepsy was chosen because mice develop spontaneous recurrent interictal spikes and seizures, as well as display many of the structural network reorganizations that reproduce the human pathology. We first used an in vitro imaging approach to study the functional reorganization of neuronal microcircuits in the dentate gyrus of the epileptic hippocampus. We found that interictal-like events were composed of the co-activation of spatially localized neuronal assemblies and that variable subsets of these clusters participated in sequential events (Feldt Muldoon et al., PNAS 2013). Thus, although these synchronous events look similar at the large-scale network level, they are actually highly variable at the level of individual neurons.To better understand epileptic dynamics in a more realistic in vivo model, we next used imaging techniques to observe spontaneous epileptiform activity in awake, chronically epileptic, head restrained mice that are free to run on a treadmill. In this setting, we have been able to determine that GABAergic interneurons are the main participants in hippocampal activity during interictal bursts (Feldt Muldoon et al., submitted), which contradicts the currently accepted view that such synchronizations are the result of runaway excitation produced from principal cells.Finally, in order to understand the relationship between early-born hub neurons and epileptiform activity, we performed immunohistochemistry on tissue slices obtained from chronically epileptic mice in which early-born GABAergic or glutamatergic neurons have been selectively labeled with GFP. We found that although much cell death occurs during the course of epileptogenesis, these early born neurons survive and future work will focus on characterizing their role in epileptiform activity in using in vivo imaging techniques.Taken together, these findings provide valuable insight into the micro-scale epileptiform dynamics that underlie macro-scale subdural EEG signals. Thus, the Hubs in Epilepsy project provides the epilepsy community with valuable information that will be essential in the development of efficient therapies to treat this disorder.