Abstracts

Rationale: The study of epileptogenesis and development of anti-epileptic drugs in vivo in animal models is cost and labour intensive. While valuable insight has been gained from in vitro studies using acute slices, these studies are hampered by additional tissue damage induced by the slicing procedure as well as the limited ability of acute slices to sustain ictal (seizure-like) activity. We here describe the spontaneous development of in vivo-like epileptiform activity in organotypic hippocampal slice cultures, which becomes apparent when cultures are recorded in their growth medium. Methods: Organotypic hippocampal slice cultures were prepared from P8 Sprague-Dawley rat pups as described by Gähwiler et al. (1981). Glial growth was inhibited by a 24 hour exposure to anti-mitotic agents following 48 hours in culture. 350 µm slices were cultured in 50% Eagles basal media, 25% Hanks balanced salt solution and 25% horse serum with 1 mM GlutaMAX in roller tubes. Field-potential recordings (1 hour) were performed after 7-30 days in vitro (DIV) under perfusion with O₂/CO₂ bubbled growth-medium using a medium-filled glass-pipette placed in CA3 under IR-DIC visualization. Recorded activity was analyzed using custom written software for event-detection. Results: After 7 DIV, only multi-unit activity was recorded in slice cultures. However, after 14 DIV, prominent interictal-like spike discharges were recorded in the majority of cultures while few displayed ictal-like discharges. After 21-30 DIV, most slices displayed prolonged ictal-like discharges mixed with interictal-like activity. The incidence of ictal-like epileptiform activity as a function of slice culture age could be fitted to a sigmoidal function with ictallike activity occurring in 50% of cultures after 21 DIV, mirroring in vivo data from rats following kainic acid induced status epilepticus (Williams et al., 2009), but on a compressed time scale. Extraction and detailed analysis of single events in the field potential revealed that 25+ DIV cultures with predomintantly ictal-like activity spend ~15% of the time in a high frequency (> 2Hz) discharge state, while 14 DIV cultures with predominantly interictal-like activity only fire >2Hz ~1% of the time. Conclusions: These results suggest that organotypic hippocampal slice cultures provide an excellent model system for the detailed study of mechanisms underlying epilpsy on the single cell and local network level, with interictal spiking preceding ictal discharges during epileptogenesis. With an ictogenesis profile corresponding to in vivo recordings, organotypic hippocampal slice cultures also offer a cost and labor effective model system suited for pharmacological testing.