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INTRODUCTION:Despite surgical resection of a defined epileptogenic focus (EF), about 50% of patients go on to have refractory seizures. Recently, focal epilepsy is being redefined as a network disorder and studies have demonstrated that resection of nodes outside the EF may contribute to better surgical outcomes. Additionally, our group has shown higher interictal connectivity between nodes involved in seizure onset in a cohort of epilepsy patients, further suggesting the presence of an underlying distributed epileptogenic network in focal epilepsy.METHODS:Primary mixed cultures of cortical neurons from P1 rat pups were cultured on 96-well multi-electrode array (MEA) plates with eight active electrodes per well for stimulation and recording. We developed an in vitro stimulation model of cortical epilepsy to study epilepsy network formation using a biphasic tetanic stimulation at 50 or 100 Hz, 500 μV, 250 μA applied for one hour daily for ten days.RESULTS:Four-days of daily in vitro stimulation demonstrated significant increases in neuronal baseline bursting, modeling kindled seizures. Tetanic stimulation with 50 Hz had the highest elevation in neuronal busting. In order to compare distributed network connectivity, we calculated and ranked coherences (13-32 Hz) across all electrode pairs (n = 24) and identified the top 15% for comparison to control. The average coherence across the top connected pairs was significantly higher in stimulated wells compared to controls (0.27 vs. 0.17, p = 0.0002). Furthermore, coherence across the highest-ranking electrode pair was strengthened with daily stimulation. This was not seen in control wells.CONCLUSION:Similar to human data, this in vitro model suggests an elevated interictal coherence among electrode pairs involved in seizure formation. Next, we aim to characterize the structure of this network quantifying axonal boutons and dendritic spines. Ultimately, our work aims to enable a better understanding of network formation in epilepsy that will inform future epilepsy surgery decision making.