Abstracts

Rationale: The rat Pilo-SE model has been utilized to study both acute effects and as a model of remote symptomatic epilepsy. The acute SE activity is typically followed by a “quiescent” period of 10-14 days after which spontaneous, recurrent seizure activity is observed. Our research group has previously shown that SE induces an acute increase in calcineurin (CaN) activity which results in loss of dendritic spines (Kurz et al 2008, Epilepsia). We have hypothesized that the SE-induced spine loss, plus reactive synaptogenesis, may be an underlying mechanism of SE-induced recurrent seizure activity. This study was undertaken to determine the temporal profile of restoration of CaN activity and dendritic spine density following SE. Methods: SE was induced in animals by injection of pilocarpine nitrate (350 mg/kg, i.p.) and monitored electrographically as routinely performed in the laboratory. Seventy min post first electrographic seizure, ictal activity was terminated by multiple diazepam injections (4 mg/kg). Animals were allowed to recover for specific periods of time after which brain tissue was harvested. CaN activity was measured by dephosphorylation of p-nitro-phenyl phosphate or Western analysis of phosphorylation state of the endogenous substrate, cofilin. In a parallel set of experiments, animals were perfused and brain tissue stained by the rapid Golgi method. Results: SE resulted in significant activation of CaN activity, as measured by biochemical assays and tracking endogenous substrates. The SE-induced increased CaN activity was observed up to 2 days post SE for both basal (160.2 +/-20% control) and maximal (140.5 +/- 10% control) activity. The elevated basal activity suggests that SE results in a long-lasting post-translational modification of the enzyme. CaN activity returned to control levels by the time animals entered into the recurrent seizure phase (14 days post SE)(basal 93.5 +/- 9.3%, maximal 89.9 +/- 4.0% p > 0.5, n = 4 each group, ANOVA). The restoration of CaN activity was confirmed by tracking the phosphorylation state of cofilin. By two weeks, the decrease in phospho-cofilin levels had restored to control levels (110.2 +/-8.96% control, p> 0.05, n = 4, Student’s t Test). Together, the data demonstrate the re-establishment of normal CaN activity as measured by both enzymatic and endogenous measures. As would be expected, the restoration of CaN activity was accompanied by an apparent restoration of dendritic spine density to near control levels in both cortex and hippocampus. Conclusions: This study characterized the sub-acute changes in CaN activity and spine density following SE in the rat. The data demonstrate the restoration of CaN activity and spine density that occurs as the animals entered the chronic, recurrent seizure activity phase. Together with previous studies, the data demonstrate a cellular mechanism through which SE may induce recurrent seizure activity, or epileptogenesis.