Abstracts

Rationale: Organotypic hippocampal cultures spontaneously develop epileptiform activity, including ictal events, after approximately 2 weeks in vitro. Compressed time scale of epileptogenesis and ease of high-throughput experiments are features that make this model of epilepsy highly suitable for antiepileptogenic drug target discovery. However, organotypic cultures are maintained in an artificial environment (culture medium) which contains salts, glucose, and amino acids that are not present at same concentrations in cerebrospinal fluid (CSF). Therefore, there is a possibility that epileptogenesis in organotypic cultures is artificially influenced by these components. We carried out a series of experiments to test the influence of medium components on epileptogenesis, and to develop a culture medium that closely approximates the composition of CSF.Methods: Slices of 350 µm thickness were dissected from postnatal day 7 Sprague-Dawley rat pups, and placed onto poly-D-lysine coated glass cover slips in 6-well tissue culture plates. Slices were maintained in various culture media (always supplemented with bovine serum albumin, insulin, selenium, glutaMAX and gentamicin) at 37 degrees C in 5% CO2 on a rocking platform. Medium was changed twice a week. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (markers of ictal activity and cell death, respectively) in culture medium, immunohistochemistry (antibodies to neural specific marker, NeuN) and automated 3-D cell counts, and extracellular recordings from CA3 and CA1 regions at different days in vitro (DIV).Results: Commercially available medium for culture of postnatal neurons, Neurobasal-A (Life Technologies), contains essential and non-essential amino acids, vitamins, glucose and salts. First, we created our own medium that replicated concentrations of amino acids in NeurobasalA but changed glucose and salt concentrations to match those found in cerebrospinal fluid. We found that this medium was capable of supporting organotypic hippocampal cultures at least as well as Neurobasal-A, but with slightly reduced ictal activity. However, ictal activity was still present in most cultures by 14 DIV. We then examined the role of amino acids. We found that reduction of essential amino acid concentrations resulted in increased cell death in organotypic cultures. On the other hand, reduction in non-essential amino acid concentrations led to slightly reduced ictal activity and reduced activity-dependent cell death.Conclusions: Some culture media (based on CSF salts and with reduced non-essential amino acids) slightly reduced ictal activity in organotypic hippocampal culture model of epilepsy. However, epileptogenesis still occurred in any culture medium that was capable of supporting neural survival, with ictal activity detected in most cultures by 14 days in vitro.