In vitro alternatives to animal models for screening and evaluation of anticonvulsive compounds

In vitro models of pharmacosensitive and pharmacoresistant epileptiform activity have been developed on the basis of organotypic slice cultures of hippocampus. Slice cultures (on average 25/animal) are prepared from 6 to 10-day old rats and cultured under 5% CO2. Single neuron and neuron population activities as well as extracellular K concentrations are recorded with microelectrodes and ion sensitive electrodes, respectively. The drugs tested were carbamazepine, phenytoin, sodium valproate, phenobarbital sodium, diazepam, clonazepam, gabapentin and ethosuximide. Drug concentrations in vitro corresponded to upper therapeutic-low toxic ranges in the brain extracellular fluid of patients treated with the respective antiepileptic drugs. High frequency stimulation of Schaffer collaterals in CA1 of hippocampus induces a primary afterdischarge (PAD) in the hippocampus. The PAD consists of a tonic and clonic component, lasts between 10–30 s and can be interpreted as an electrographic correlate of the maximal electroshock seizure (MES) test in animals. The pharmacosensitivity of the PAD is similar to that of the MES-Test: the PAD is inhibited by phenytoin and carbamazepin and not by ethosuximide. 1,4benzodiazepines and sodium valproate cause a partial suppression of the PAD only. Low magnesium or blockade of Kchannels reliably induces recurrent tonicclonic seizure like activity in the hippocampus and the dentate gyrus. In more than 94% of 120 slice cultures investigated so far the studied antiepileptic drugs failed to block induction of epileptiform activities even when toxic concentrations were applied. The pharmacoresistance persisted over the time in vitro explored so far (2 months). We present here two simple to establish in vitro models, one of pharmacosensitive, the other of pharmacoresistant epileptiform activity of which the latter in comparison to respective animal models is a priori pharmacoresistent. After further validation the in vitro models might be well suited for inclusion in the panel of tests used for the identification of new antiepileptic compounds thus reducing the number of animals currently used in anticonvulsive drug screening and testing and eventually replacing the respective animal models.