Abstracts

Rationale: Epileptogenesis of a neuronal circuit develops gradually and includes loss of inhibition and resultant hyperexcitability. Reorganization of neural circuitry is also suspected. Here, we used multi-electrode arrays to record activity from multiple neurons across excised hippocampal circuits in chronic epileptic (pilocarpine-treated) and normotypic control rats and applied a graph theory-based network analysis to quantify both global-level (whole network) and local-level (single electrode) activity.Methods: Male Sprague Dawley rats (~ 40 days old) received atropine methyl nitrate (2.5 mg/kg s.c.) followed by pilocarpine hydrochloride (380 mg/kg s.c.). Diazepam (5mg/kg i.p.) was administered 1 hour after the onset of status epilepticus, or 3 hours after pilocarpine injection if no status occurred. Control animals were treated identically but received saline (3 ml/kg) instead of pilocarpine, or received pilocarpine, but did not develop status. A 100-position Utah-type multielectrode array was lowered onto slices prepared from pilocarpine-treated and control rats. The platinum-coated tips (0.4 mm pitch) project 1mm from the silicon substrate and can be positioned at best depths to record from a viable region. Recordings were made from 96 electrodes at 30 KHz/channel. The array was lowered further after recording to puncture the slice for staining to confirm location of individual electrodes. Neuronal event (NEV) files were processed to identify putative connected neurons using a specified moving time window consistent with a monosynaptic connection (3-10 msec delay). An edge list was made for each neuron pair that fit this criterion using R and network metrics applied to the subsequent output with CASOS ORA software. Results: Slices from 23 animals (7 control, 16 epileptic) were compared. Both epileptiform burst discharges and intra-burst activity were observed. Overall measures of network density indicate that the number of links did not differ significantly between epileptic and control slices. However, fewer neurons were responsible for making more connections in the chronic epileptic tissue. This was demonstrated by the proportion of neurons that showed a high betweeness centrality (equal or greater to the mean) compared across groups. Neurons with high betweeness centrality likely represent connective hubs that may increase synchronization within the circuit. Chronic epileptic rats had a significantly increased number of these high betweeness nodes (65% vs 40% in controls, p> 0.05). Correlation of anatomical locations with electrodes suggests the presence of aberrant back projections within the circuit. Future measures will determine if the back projections are directed towards hub neurons.Conclusions: Network analysis provides a useful toolbox to measure global and local abnormalities in health and diseased neuronal networks. Further measures such as relative reciprocity will yield a better understanding of hippocampal reorganization in epileptogenesis.