Abstracts

Enhanced hippocampal excitability after experimental prolonged febrile seizures has recently been correlated with alterations of HCN channel expression (Brewster et al., 2002). Since HCN changes may provide a mechanism for seizure-provoked epileptogenesis, understanding how seizure-like activity regulates HCN expression is highly desirable. Studies of HCN regulation [italic]in vivo[/italic] are constrained by the complexitiy of the system; therefore, we chose to conduct experiments in the organotypic slice culture.
To approximate the developmental time-frame of febrile seizures, organotypic slice cultures were prepared from P8 rats amd cultured for up to 14 days (Stoppini et al., 1991). Epileptiform activity of differing duration was evoked using a low [Mg²⁺]-medium (0.4 mM), and neuronal activity patterns were monitored using extracellular recordings and c-fos-immunocytochemistry (ICC). Expression of HCN1 and HCN2 mRNA and protein was determined using [italic]in situ[/italic] hybridization and ICC, respectively.
HCN1 and HCN2 mRNA and protein levels were robust under normal medium conditions, and generally followed the patterns observed [italic]in vivo[/italic]. For both isoforms, mRNA signal was strong in pyramidal cells and interneurons, with HCN1 more prominent in CA1, and HCN2 signal stronger in the CA3 pyramidal cell layer. As[italic] in vivo[/italic], HCN1 immunoreactivity was pronounced in dendrites, whereas an antibody for HCN2 labeled preferentially cell bodies. Low [Mg²⁺] reliably evoked epileptiform activity that was associated with increased HCN2 (p = 0.07) in CA1 and CA3a. Changes of HCN1 mRNA expression were modest.
HCN1 and HCN2 expression in the organotypic slice culture reproduce [italic]in vivo[/italic] patterns. Importantly, our finding that activity modulates channel expression [italic]in vitro[/italic] enables future studies of the mechanisms involved.
[Supported by: NS 35439; NS 28912 (TZB, ALB); EFA/Milken Foundation (RAB)]