Abstracts

Rationale: Neurogenesis persists throughout adulthood in the hippocampal dentate gyrus (DG) and is stimulated by seizures. In rodent temporal lobe epilepsy (TLE) models, dentate granule cells (DGCs) born after status epilepticus (SE) develop aberrant connections and may contribute to pathophysiology. To examine how SE affects the integration of newborn DGCs into the pre-existing circuitry, we utilize a dual-virus approach to identify afferent inputs onto neonatally- or adult-born DGCs. A retrovirus (RV-CAG-GTR) expressing green fluorescent protein (GFP), an avian viral envelope protein receptor (TVA), and rabies glycoprotein (Rgp) driven by the CAG promoter is used to selectively infect dividing cells in the rat DG. An avian envelope protein (EnvA)-pseudotyped rabies virus, with Rgp replaced by mCherry (RbV-mCh), is subsequently introduced into the same DG to selectively infect RV-CAG-GTR transduced neurons through EnvA binding to its cognate receptor, TVA. RbV-mCh complements with Rgp provided by RV-CAG-GTR and retrogradely crosses synapses, labeling first-order presynaptic neurons with mCherry. Because the traced presynaptic neurons lack Rgp, the RbV-mCh virus does not spread further. We used this approach to examine presynaptic inputs onto neonatally- vs. adult-born DGCs in rats undergoing pilocarpine-induced SE.Methods: We stereotaxically injected RV-CAG-GTR into postnatal day (P) 7 or P60 rat DG. SE was induced with pilocarpine at P56. At 10 wks after SE, animals received bilateral DG injections of RbV-mCh to retrogradely label presynaptic DGC inputs and were euthanized 7 d later. Controls received saline instead of pilocarpine. We identified the starter neurons by dual expression of GFP and mCherry. Presynaptic neurons innervating starter neurons express mCherry only. Results: Retrograde trans-synaptic labeling was seen from starter DGCs to other DGCs as well as hilar, DGC layer and molecular layer interneurons in both SE and sham treated animals. We observed the most mCherry+ cells in SE animals with P60 RV injections and the fewest in SE animals with P7 RV injections. This result was unexpected, as mature DGCs should have more presynaptic connections than newborn DGCs, which have not had as much time to integrate into the neuronal network. Sham-treated animals had intermediate levels of mCherry+ cells. We did not see mCherry expression in entorhinal cortex with dorsal DG injections. Conclusions: These preliminary findings suggest that connections onto DGCs born after SE are different from those onto DGCs that were mature at the time of SE. These differences include the amount and type of presynaptic inputs, with more DGC inputs onto adult-born DGCs. Lack of labeling in entorhinal cortex in epileptic and control rats may relate to the long distances for retrograde labeling. Ongoing studies are aimed at identifying the different presynaptic cells, and determining whether double-labeled DGCs are starter cells vs. retrogradely labeled cells synapsing onto other DGCs. Grant Support: NIH NS058585.