Abstracts

Rationale: Brain insults such as ischemia or infection produce excess excitation and neurotransmitter release and may result in subsequent epilepsy. 4-aminopyridine (4-AP), a potassium channel antagonist, causes hyperexcitability as well as elevated transmitter release. I therefore investigated the effects of growing hippocampal slice cultures in media containing 4-AP. Methods: Organotypic hippocampal slice cultures were prepared from postnatal day 6 C57/B6 mice and grown in standard media with or without 200 micromolar 4-AP. After 5 to 11 days in vitro (DIV), extracellular CA1 field recordings were obtained while perfused with artificial CSF. In “washout” experiments, cultures were grown in media with 4-AP for 6 DIV then in normal media for another 1 to 5 days. Results: Prolonged repetitive epileptiform discharges were recorded in area CA1 from 19 of 20 cultures grown in 4-AP (4-AP cultures) but in only 1 of 20 grown in normal media (control cultures). Washout experiments showed persistence of the hyperexcitability for up to 5 days. Quantitative immunoblots revealed significant increases in glutamic acid decarboxylase (GAD) and vesicular GABA transporter (vGAT) expression levels in slice cultures grown in 4-AP. Although such a change appears compensatory, the slice cultures grown in 4-AP also demonstrated a relative decrease in expression of the KCC2 chloride cotransporter and a relative increase in expression of the NKCC1 chloride cotransporter as compared to cultures grown in control media. Such a change would tend to prolong the time period during which GABA-A responses are depolarizing. Conclusions: Growing hippocampal slice cultures in media containing 4-AP produces persistent hyperexcitability which is associated with increased expression of GABA synthetic and vesicular packaging proteins as well as maintenance of an immature chloride cotransporter phenotype. These results suggest that compensatory upregulation of inhibition may exacerbate hyperexcitability in the immature brain. Such findings illustrate potential pathogenic mechanisms in developmental epilepsy as well as possible targets for pharmacologic intervention. Supported by: NIH NINDS NS18309, NS43124, and K08NS054882.