Abstracts

Rationale: In infants, severe traumatic brain injury involving extensive axonal shearing leads to epilepsy in up to 60% of patients. Presumably, homeostatic restoration of neural inputs in brain tissue that has been isolated by injury leads to excess recurrent connectivity, positive feedback loops, and a neural circuit prone to seizure. But, functional evidence of such pathological neural re-wiring remains elusive.Methods: Here, we model pediatric post-traumatic epilepsy using organotypic slices, which are prepared from neonatal mice (P5-P8) using shearing trauma (slicing). Slices become severely epileptic over the course of 3 weeks in culture. The mice used for these experiments express a floxed, FRET-based calcium sensor Yellow Cameleon 3.6 (YC3.6) under the control of Nestin-Cre, resulting in chronic expression of YC3.6 in neurons, glia, and blood vessels. Culturing the brain slices in a sealed petri dish with a cover slip bottom enabled repeated, sterile imaging of the same slice cultures. Using two-photon, targeted path scan (TPS) imaging, we recorded 30 minute epochs of spontaneous activity from at least 30 cells at a sampling rate of 25-50Hz (sampling rate with TPS depends on the path selected). These imaging sessions were repeated at consecutive weeks in vitro, as the slices underwent epileptogenesis. To quantify changes in neuronal population dynamics, we used network analysis techniques in which nodes on a graph represent neurons and connections are drawn based on cross-correlation strength between calcium signals. The degree of local connectivity was quantified for the network by computing the clustering coefficient , the percentage of connections among nodes with a connection to a common node.Results: TPS calcium imaging reveals a pattern of epileptogenesis consistent with previously published electrophysiological measurements, with epileptiform activity appearing as large, spatially correlated increases in calcium (manifest as increases in the YC3.6 ratio). Preliminary data show that the average clustering coefficient in both CA3 and CA1 continuously increases throughout epileptogenesis during the first three weeks in vitro.Conclusions: We hypothesize that reconnection of severed axons to a limited number of target neurons produces a hyper-connected, epileptic neuronal network. While the slicing used to produce organotypic cultures represents an extreme example of traumatic brain injury (resulting in epileptogenesis in 100% of hippocampal slice cultures), we propose that axonal sprouting following injury in vivo fosters epilepsy by a similar principle.