Abstracts

Rationale: EEG spikes and seizures coexist in epileptic patients. These two types of discharge differ in frequency and duration, but the mechanisms underlying these differences, and the role of these activities in epileptogenesis is unknown. Here we use electrical recordings and calcium imaging in organotypic hippocampal slice cultures to investigate differences in the onset pattern of these two types of activity in control conditions and after blocking synaptic inhibition. Methods: We used high-speed calcium imaging of organotypic hippocampal slice cultures, which were sufficiently thin to permit imaging of the entire CA3 network without confocal or 2-photon technologies. Nearly 100% of these cultures develop both ictal- and interictal-like activity after 3 weeks in culture. Spontaneous epileptiform activity was imaged in Hanks Balanced Salt solution with 1 mM Glutamax. We also looked at the onset of epileptiform activity after eliminating synaptic inhibition with 100 uM picrotoxin + 1uM CGP 55845. Electrical fields were recorded using a tungsten electrode to verify the presence of interictal and ictal activity. Calcium transients associated with action potentials and paroxysmal depolarizing shifts of the membrane potential were imaged using AM dyes (e.g. Oregon Green Bapta-1, Molecular Probes). Slow glial signals were differentiated from neuronal signals by high-pass temporal pixel filtering. Neuronal calcium signals were mapped onto a Cartesian coordinate system. Propagation of calcium transients was quantified using 3-D bubble plots in which bubble diameter represented the area with increased calcium concentration; x,y coordinates represented the median x,y positions of all such neurons within the network; and the z axis represented time. These analysis techniques allow us to distinguish neuronal vs. glial contributions to the propagation of activity. Results: We compared the geometry (onset and ignition trajectory) of bursts and seizures in each culture and compared the variance before and after blocking inhibition. We demonstrate distinct patterns of initiation and spread of activity for these two types of epileptiform discharges. Our data suggest substantial variance in the onset of interictal bursts that is diminished by pharmacological disinhibition. Propagation speed is increased by disinhibition. Seizure initiation appears to be distinct from interictal spike initiation in both onset and spread of activity, although the relative rarity of seizures vs. spikes necessitate additional data collection. Conclusions: In organotypic hippocampal networks, seizure-like and interictal-like bursts of network activity generate elevations in neuronal calcium that have different onset kinetics and patterns of spatial propagation. These findings provide a new avenue of investigation into the differences between these two fundamental types of epileptiform discharge. The different calcium dynamics accompanying these forms of activity likely influence long-term plasticity of the CA3 network and contribute to epileptogenesis.