Abstracts

Rationale: Folowing traumatic brain injury, a latent period of neural repair, involving sprouting and synaptogenesis, often results in epilepsy. However, some of the most basic patterns of this neural re-wiring and their relation to the dynamics of the network remain unknown. Understanding the neuronal response to injury and the tipping pointing between physiological neural repair and epileptogenesis will be critical in designing prophylactic treatments for post-traumatic epilepsy. Methods: Here, we use the experimentally accessible organotypic hippocampal slice culture model of post-traumatic epileptogenesis. Following the trauma of slicing, the cultures spontaneously and progressively become epileptic, generating interictal-like bursts in the first week and a combination of interictal spikes and seizures during the second week in vitro and beyond. We either prepared slices from transgenic animals or infected slices with AAV vectors to achieve stable expression of genetically encoded calcium indicators. Cultures were incubated in optically accessible cover slip-bottomed petri dishes to permit longitudinal imaging, while maintaining sterility. Two-photon targeted path scan imaging was used to periodically image activity in populations of 30-40 cells during the transition from silence to bursting and seizure. Functional network connectivity was quantified using correlation-based network analysis of calcium traces.Results: We found that statistically significant correlations are initially weak and only exist between nearby pairs of cells. As interictal bursting continued, these correlations became progressively stronger and more far-reaching, with correlation strength between pairs of neurons becoming nearly independent of the distance between them. The emergence of long-distance correlated activity coincided with the onset of long-duration seizures. Interestingly, in this severe model of injury, bursts of synchronous activity began within hours of slice trauma. To more closely examine network changes associated with the initial emergence of synchrony after injury, slices were cultured in a stage-mounted miniature incubator and the same 40 cells were imaged hourly for the first 24 hours in vitro. During this time, a similar pattern of initially local and progressively more distal correlations was observed.Conclusions: These data support a model in which neurons replace synapses lost to injury by connecting first to neighboring cells, and indicate that the emergence of seizure activity is coincident with the development of longer-range connectivity.