Abstracts

Rationale: Status Epilepticus (SE) is a life-threatening emergency affecting over 100,000 people annually in the United States and is associated with a significant mortality rate. While numerous alterations at the molecular level have been documented during the progression of SE, molecular mechanisms that precede the onset of SE have yet to be elucidated. This study examined the modulation of CaM kinase II activity prior to SE onset. Methods: Spectral analysis of ictal activity during the discrete seizure phase was performed in the pilocarpine rat model. Specific electrographic bins of animals with known fates (SE or SZ-no SE) were utilized to construct the linear discriminant function (Constant = -.5 Xj cov-1Xj coefficient vector = cov-1Xj). The posterior probability of membership into each group was estimated according to the equation: Pr(jlx)=exp(-.5D2j(s))/sumkexp)-.5D2k(x). A cross-validation was performed using the jack-knife method in which one observation was predicted using the discriminant function constructed from the remaining set. In a parallel experiment, brain tissue was isolated at specific time points after the first discrete seizure and CaM kinase II activity characterized by enzymatic reactions and quantification of AMPA . Subcellular fractions were isolated as routinely performed in the laboratory. Results: Discriminant analysis of the first discrete seizure in animals of known outcome was able to predict the SE and non-SE groups (n = 22). Inclusion of 5 spectral bins, delta, theta, alpha, beta1, and beta2 predicted inclusion in either SE or SZ-no SE with a 0% error rate. Cross validation using the jack-knife method demonstrated that the discriminant function was 100% specific—each subject was assigned to the correct group 100% of the time. In the next experiment (n = 19), brain tissue was isolated at 6 min (5:55 +/-13 sec, n = 5) and 8 min (8:35 +/- 25 sec) following the first observed discrete seizure. Previous studies have demonstrated that SE onset occurs approximately 9 +/- 1 min post-first seizure. Discriminant analysis estimated the 6 min group had a 94.2 + 4% and the 8 min group had a 99.6 + 0.4% probability of reaching SE. A significant inhibition of total kinase activity was observed at all points tested (79% and 50.3% control activity). In contrast, there was an increase in CaM kinase II activity and enrichment of enzyme expression in the crude synaptic membrane (SPM) fraction (106.7 and 121.0 % control activity). In addition, a significant increase in GLuR1 phosphorylation was observed (134% and 167% control). Conclusions: The results of these studies demonstrate the ability to predict the probability of progression to overt SE by discriminant analysis of the first discrete seizure. Utilizing this powerful tool, data was generated that demonstrated a significantly increased CaM kinase II-dependent phosphorylation of AMPA channels that preceded SE onset. The increased phosphorylation may be a mechanism through which pilocarpine induces excessive excitatory drive that may overwhelm the innate mechanisms to terminate seizure activity in the model.