Abstracts

Rationale: A seizure-prone neural circuit may form following an insult to the brain (e.g. post-traumatic epileptogenesis). The spatial organization of seizure activity and the evolution of seizure propagation patterns throughout epileptogenesis remain relatively unknown.Methods: We prepared organotypic slice cultures from DLX-cre mice infected with AAV vectors to achieve stable expression of genetically encoded calcium indicators in all neurons and red fluorescent protein in interneurons only. Cultures were incubated in optically accessible cover slip-bottomed 6-well plates, equipped with media perfusion ports, to permit longitudinal imaging, while maintaining sterility. We modified a CO2 incubator to include optics for inverted fluorescence microscopy, enabling us to chronically image activity in up to 6 slice cultures over the course of weeks, as the slices became epileptic. At four hour intervals, anatomical z-stacks and 4-minute movies of calcium dynamics were acquired to characterize interneuron distribution and propagation of epileptiform activity respectively. Furthermore each slice was recorded daily for a 4-hour epoch using a low-light “optical field potential” (OFP) to quantify seizure frequency and duration.Results: OFP recordings reflect a pattern of epileptogenesis previously demonstrated in organotypic slices using acute and chronic electrophysiological recordings: interictal spikes develop in the first 2 days in vitro (DIV), followed by the emergence of seizure-like events. During the first 5 days following initial seizure onset, seizures gradually become longer in duration and more frequent, with progressively less interictal activity. For each 4-minute recording of calcium activity, we extracted calcium traces for individual principal neurons and interneurons (identified by expression of tdTomato). We found that, at seizure onset, stratum oriens interneurons consistently fired before CA1 pyramidal neurons. This pattern of activation persisted for >1week of seizure activity, despite significant changes in seizure duration and pre-ictal buildup.Conclusions: In a preparation in which all active neurons are directly visualized, we provide evidence at the cellular level that ictogenesis continually evolves as the network re-wires following injury. Preliminary data indicate that throughout epileptogenesis, activity in s.oriens interneurons reliably precedes CA1 pyramidal neuron activation during seizure onset.