Abstracts

Rationale:
Acquired epilepsies are characterized by spontaneous, recurrent seizures that emerge following injury. Understanding seizure initiation, or ictogenesis, is critical for developing treatments for medically refractory epilepsy. However, ictogenesis is difficult to study, due to the spontaneous nature of seizures and poor spatial sampling available for capturing seizure onset in vivo. Here, we developed novel techniques for chronic cellular resolution imaging of neuronal activity in the hippocampal organotypic slice culture model of post-traumatic epilepsy.
Method:
Hippocampal slice cultures generate spontaneous seizure-like events following slicing injury. With this preparation, it is feasible to image the entire network, ensuring that the earliest pathological activity is captured (as no outside inputs exist). Pan-neuronal GCaMP7 expression was paired with DLX-cre-driven tdtomato labeling of interneurons to separate the calcium activity of principal cells and interneurons.  We used a custom imaging system to observe undisturbed organotypic hippocampal slice cultures over several weeks to capture the calcium activity of hundreds of individual neurons, for dozens of seizures. We used this preparation to test whether a subpopulation of neurons consistently initiates seizure activity.
Results:
Electrophysiological recordings and calcium imaging demonstrated 3 unique types of seizure onset in this preparation: low amplitude fast, hypersynchronous and spiking onset. Over the course of epileptogenesis, seizure onsets demonstrated consistent evolution to more synchronous forms. Whole-network, cellular resolution imaging revealed that seizure onset is characterized by widespread, moderate increases in activity among many neurons, (rather than high activity among a small number of cells). We further found that interneuron activity precedes that of principal cells during ictogenesis, but to a relatively modest degree. We utilized our cellular level tracking to test if a discrete population of cells was repeatedly active at seizure onset. Although the evolution of hippocampal regional onset was fairly consistent, we found that neuronal onset sequences were highly variable seizure to seizure.
Conclusion:
These results suggest that seizure onset is stochastic at the cellular level and argues against the presence of a small number of neuronal drivers that initiate seizure. Transition to seizure onset may therefore be driven by widespread state changes that allow for re-entrant seizure activity over non-unique synaptic circuits.  
Funding:
:NIH