Abstracts

Rationale: Organotypic hippocampal slices cultures (OHSC) have been developed as an in vitro epilepsy model and are considered to reflect epileptogenesis and chronic seizures. OHSCs have been used to validate involvement of a pro-inflammatory cytokine in epileptogenesis, including confirmation of antiepileptic efficacy of an antagonist for CSF-1R identified with a systems genetics approach integrating transcriptomic data from pilocarpine mouse hippocampus and human temporal lobe epilepsy (hTLE) samples. To characterize the molecular relevance of the model and translational value for hTLE, we use this approach on OHSCs to identify transcriptional regulatory changes in the course of epileptogenesis in OHSCs and compare these to regulations previously identified in pilocarpine mouse and hTLE. Methods: Hippocampal slices were prepared from 7-9 day-old rats and cultured on multi-electrode arrays (MEAs) for ~21 days in vitro (DIV). The phenotypic epileptic readout was obtained through an electrophysiological evaluation using local field potential (LFP) recordings at either ~7 DIV, ~14 DIV or ~21 DIV. Then the slices were frozen and processed for RNA sequencing. Differential expression analysis used the Bioconductor package EdgeR. Co-regulation analysis was performed to identify new gene co-expression modules. Enrichment analysis was performed to functionally annotate the modules and their association to epileptic phenotype and time in culture was assessed. Conservation of the modules in pilocarpine mouse and hTLE was determined by comparing the median R² to the null distributions. Results: LFP recordings from validated OHSCs (n=87) on MEAs confirmed spontaneous development of epileptiform activity consisting of ictal-like and interictal-like activity in the model. The ictal activity was already observed at DIV7 in 48% of the slices and continued to develop progressively before reaching 60% of the slices at DIV14 and finally 82% of the slices at DIV21. Co-regulation analysis of the OHSC samples identified 17 modules which were annotated according to their biological function. Amongst the 17, 6 modules are of particular interest due to their conservation of regulation in pilocarpine mouse and hTLE, their association to the epileptic phenotype, and change in regulation over time, suggesting candidate causal and disease-relevant mechanisms reproduced in OHSCs. Conclusions: Our findings revealed that several co-regulation modules identified in the in vitro OHSC model correlate with the progression of the ictal phenotype and are conserved in mouse and human epilepsy datasets. This implies that OHSCs can be considered as a translational model for hTLE. Future investigations using a causal reasoning analytical framework for target (CRAFT) discovery approach will help to identify novel gene regulators involved in hTLE. Funding: UCB Pharma-sponsored