Abstracts

Rationale: Seizures are generally thought to arise from a shift from balanced excitation and inhibition to increased excitation. However, recent genetic studies of human epilepsy demonstrate loss of function mutations in voltage-gated Na2+ channels that presumably disrupt excitatory mechanisms. Similar loss-of-function mutations occur in cardiac Na2+ channels, and reduce the conduction velocity of excitatory transmission. When the rate is sufficiently low, propagating circular waves of excitation return to their origin following recovery from a refractory state, and pathological reentrant activity occurs. Repeated activation of excitatory pathways may occur during seizures in patients with Na2+ channel mutations, and this may account for the local rhythmic EEG activity that precedes focal seizures and the activity-dependent degradation in inhibition that leads to the spread of seizure activityMethods: To test the feasibility of this “cerebral fibrillation” hypothesis of epileptogenesis, we combined electrophysiological recordings with calcium imaging of hippocampal CA3 organotypic slice cultures. We used the roller-tube technique to obtain the thinnest possible cultures, which allows entire networks of CA3 neurons to be imaged simultaneously. Calcium transients associated with epileptiform-related paroxysmal depolarizing shifts were imaged with AM dyes (eg Fluo-4), slow glial calcium signals were removed, and neuronal signals were mapped onto a Cartesian coordinate system. Propagation of epileptiform activity was quantified using 3-D bubble plots: bubble diameter represent the number of active neurons; x,y coordinates represent median x,y positions of all neurons within the network; and the z axis represents time. Propagation pathways were compared under control conditions (100 uM picrotoxin/ 1uM CGP 55845), and after loss of function modeled by 1) reducing synchrony of glutamate release by substituting extracellular calcium with strontium and 2) reducing sodium conductance (nanomolar concentrations of TTX). Results: Organotypic CA3 slices disinhibited by picrotoxin/CGP displayed epileptiform activity that was recorded extracellularly. Altering excitatory transmission changed the pattern of burst propagation visible with calcium imaging. Strontium increased the duration of CA3 epileptiform discharges.Conclusions: Reduced excitatory mechanisms (eg synchronous glutamate release) can prolong epileptiform discharges, likely due to 1) a reduced rate of onset of the synaptic depression that typically terminates CA3 bursting and 2) reduced conduction velocity allowing waves of excitation to return to the area of origin after recovery from a refractory state 3) reduced fidelity of axonal conduction (ie more failures in TTX). Our modeling studies have demonstrated spiral waves of excitation consistent with reentrant patterns of neuronal excitation, and here we report calcium-imaging studies testing whether corresponding patterns of activation are observed in synaptically coupled neurons.