Abstracts

Rationale: We have recently found that the onset of spontaneous seizures in the pilocarpine model of epilepsy causes a hyperpolarized shift in the voltage-dependent activation of hyperpolarization-activated cation (HCN) channel-mediated current (Ih), associated with increased neuronal excitability. We also showed that activation of p38 mitogen-activated protein kinase (p38 MAPK) or inhibition of the phosphatase, calcineurin (CaN) upregulated Ih voltage-dependent activation under normal conditions. An in vitro model of seizures, in which perfusion for one hour with 0 Mg2+ and 50 μM bicuculline at 35 - 37°C, caused spontaneous seizure-like events in slices and also hyperpolarized Ih activation. Following the seizure-provoking conditions, FK506 (an inhibitor of CaN) reversed the seizure-induced hyperpolarized shift in Ih activation to control levels. These results indicate that seizure-induced loss of phosphorylation may underlie changes in HCN channel gating in epilepsy. Methods: To test the role of p38 MAPK using the an in vitro model of seizures, following the seizure-provoking conditions, anisomycin (20 μM, activator of p38 MAPK) was perfused on hippocampal slices, and cell-attached patch-clamp recordings were performed in CA1 pyramidal neuron dendrites. Further, we examined the levels of phospho-p38 MAPK with Western blotting to determine whether there is any loss of p38 MAPK activity in epileptic animals. CA1 hippocampal areas were used for the Western blotting from epileptic animals during the chronic period at 3 weeks postpilocarpine. Results: Dendritic recordings showed that the Ih half-activation voltage (V1/2) following in vitro seizures was -100 ± 2.7 mV (n = 20), and activation of p38 MAPK with anisomycin (20 μM) shifted V1/2 to -85 ± 2.4 mV (n = 8), similar to application of FK506 (-88 ± 3.6 mV, n = 8), and to control (-89 ± 1.2 mV, n = 22). However, neither p38 MAPK activation nor CaN inhibition following seizures restored Ih activation to the depolarized levels seen in control slices treated with anisomycin (-78 ± 2.1 mV, n = 7) or FK506 (-78 ± 2.8 mV, n = 8). However, co-application of FK506 and anisomycin following seizures did shift V1/2 to the levels (-80 ± 1.3 mV, n = 7) seen in control slices incubated with FK506 and anisomycin together (-81 ± 1.6 mV, n = 7). In addition, CA1 hippocampal levels of phospho-p38 MAPK in epileptic animals with Western blotting were significantly reduced (60 ± 12 % of control, n = 7, p < 0.05), while levels of total p38 MAPK were slightly increased compared with age-matched control animals (127 ± 4.9 % of control, n = 7, p < 0.01). Conclusions: These results suggest that decreased levels of phosphorylation following spontaneous seizures may downregulate HCN channel gating, mediated in part by reduced p38 MAPK activity, and possibly by increased CaN activity as well. Because decreased HCN channel activity exerts a pro-convulsive effect, activation of p38 MAPK may produce an antiepileptic action in the pilocarpine model.