Abstracts

Rationale: In secondary epilepsy, a seizure-prone neural circuit forms following an insult to the brain (e.g. traumatic brain injury). The nature of both the epileptogenic changes that take place during this latent period and the continuing evolution that occurs following the onset of seizures remain relatively unknown. Methods: We have used both whole-cell patch clamp electrophysiology and serial two-photon calcium imaging to characterize network changes occurring in the hippocampal organotypic slice culture model of post-traumatic epileptogenesis. We 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 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: Voltage-clamp recordings revealed a progressive increase in mEPSC (n=78 recordings, p = 0.0015) and mIPSC (n=81 recordings, p = 1.05x10^-5) frequency throughout the first week in culture, suggesting an increase in the number of functional excitatory and inhibitory synapses. Calcium imaging revealed that over the first 24 hours, as interictal bursts emerged, there was a rapid increase in the proportion of neurons participating in the bursts and in the synchrony of each burst. Ictal activity typically began during the second or third DIV, and evolved more gradually. Over the first 8 DIV, seizure onset became progressively more synchronous across the population of neurons imaged. Cross-correlation-based network analyses quantitatively confirmed that, in "early epilepsy" seizures, there was a slow buildup of predominantly local, correlated activity preceding seizure onset. In "late epilepsy" seizures, there was an abrupt transition from unmeasurable to ictal calcium activity, corresponding to a sharp, spatially uniform increase in correlated activity. Concomitant with the decrease in pre-ictal buildup of activity, was a significant decrease in measurable, non-ictal calcium transients, suggesting that, in late epilepsy, moderate bursts of synchronous activity are sufficient to initiate seizures. Interestingly, despite the many changes in correlation structure and temporal dynamics in the network, the percentage of time neurons spent "active" (above the empirically determined detection threshold for our calcium indicator) remained constant (n = 5 slices, 40 recordings, 30 neurons/recording, p = 0.389). Conclusions: We provide direct evidence at the cellular level that epilepsy is a progressive disorder, in which the neural network continually becomes more densely connected. Furthermore, throughout epileptogenesis, regardless of synaptogenesis or network dynamics (random firing, bursting, seizure), neurons maintain an approximately 10% duty cycle, suggesting that homeostatic targets can be reached even in the presence of continuous seizing.