Abstracts

Rationale: Epileptogenesis, the process of development of chronic epilepsy after an initial insult such as status epilepticus (SE), is comprised of complex cellular and molecular changes. In rodents, SE (continuous seizures lasting at least over 5 minutes) triggered by pilocarpine is followed by the development of spontaneous recurrent seizures, which makes it a well-suited model to study epileptogenesis. The goal of these studies was to explore changes in translational processes in pilocarpine-induced epileptogenesis using polyribosomal profiling. Methods: Male Sprague Dawley rats (32-45 days old) received scopolamine (1 mg/kg; i.p). Thirty minutes later pilocarpine (320 mg/kg; i.p.) or vehicle (saline; i.p.) was administered. Within 1 hour the animals developed SE which was terminated by pentobarbital (30mg/Kg; i.p.). Twenty-four hours after SE, animals were euthanized and the hippocampi were collected. Whole hippocampi were homogenized in lysis buffer containing cycloheximide or puromycin. Post-nuclear extracts were layered on top of 15-45% sucrose gradient and centrifuged at 35,000 rpm for 2 hours at +4C. Sucrose gradients were fractionated using the BR-184 tube piercer and a syringe pump fitted with a UA-6 UV detector. Digital data were collected with the Peak Chart Data acquisition software. The 254 nm absorbance curve versus time was plotted and area under the curve was calculated. The polysome/monosome ratio was calculated as a ratio of areas under respective segments of the curve. Results: The fidelity of our fractionation was confirmed with puromycin release experiments, the analysis of ribosomal RNA (rRNA) occupancy and immunoblotting to detect eukaryotic initiation factor 3C (eIF3C) and ribosomal protein S6 (rpS6) in non-polysomal fractions. Visual examination of the profiles in SE (n=6) seemed to indicate a global increase in protein translation as compared to the Sham animals (n=5). However the calculation of the polysome/monosome ratio failed to show any significant difference in SE versus Sham rat hippocampi (1.03 0.36 vs. 1.08 0.07; n =5-6) indicating that overall translation was not significantly altered. Conclusions: Via polysome profiling, no significant change in global translation could be detected in a comparison of SE and Sham rats 24 hours after induction. However further analysis of the polyribosomal fractions with mRNA profiling will evaluate whether there are alterations in any sets of mRNAs being translated during epileptogenesis. We also plan to evaluate later time points following SE. Our strategy will allow us to define whether translational programs are altered following SE. Funding: NIH RO1 NS081053