Abstracts

Rationale: Axonal reorganization of dentate granule cells (DGCs) is a prominent feature of temporal lobe epilepsy (TLE). Mossy fiber sprouting into the inner molecular layer (IML) has been investigated for decades using immunohistochemistry, Timm staining, and biocytin labeling of individual cells, but little is known about axonal changes in the hilus and CA3. In the hilus, in particular, changes in synaptic connections have been inferred, but not characterized. In CA3, recent work shows seizure-induced changes in bouton size, structure, and number, but nothing is known about the identity of DGCs responsible for these changes. Findings from our laboratory and others indicate that DGC maturity during epileptogenic insults is a critical factor in how the individual cell responds to the insult and the extent to which it participates in aberrant circuitry. We therefore examined the potential relationship between DGC age and extent of seizure-induced axonal reorganization. Methods: To study axonal projections of age-defined cohorts of DGCs born before or after pilocarpine-induced status epilepticus (SE) we generated a retrovirus (RV) construct in which synaptophysin, a synaptic vesicle protein that is targeted to boutons, is conjugated to a yellow fluorescent protein (YFP). RV only integrates into dividing cells, which allows birthdating of DGCs with labeled axon terminals based on when the RV was introduced into the dentate gyrus. Male Sprague Dawley rats were subjected to the pilocarpine TLE model, undergoing SE at postnatal day 56 (P56). RV was injected into the dentate gyrus either at P7 (labeling cells that would be seven weeks old at the time of SE) or at P60 (labeling cells born 4 days after SE). Controls for either group received saline in place of pilocarpine. Brains were perfusion-fixed 10 weeks after SE/sham treatment and sectioned at 60 µm to visualize cells. Tissues were processed for immunohistochemistry using antibodies against YFP and bassoon (pre-synaptic marker), and images were acquired on a confocal microscope. P60-injected groups were analyzed first. Results: We found robust labeling of mossy fiber boutons in the hilus and CA3 in all animals, as well as in the IML in epileptic rats injected with RV 4 days after SE. In CA3, we observed increases in bouton size and in the number of satellite boutons in the P60 SE group. In the hilus, DGCs labeled after SE showed increased bouton density compared to controls. Conclusions: These findings suggest that mossy fibers of DGCs generated after SE are highly susceptible to seizure-induced plasticity in the hilus, IML and CA3. Ongoing work aims to compare DGCs generated at P7 with those born on P60, and to elucidate the post-synaptic targets of remodeled axons. Supported by an Epilepsy Foundation Predoctoral Fellowship (ALA) and NINDS NS058585 (JMP)