Abstracts

Dentate granule cell (DGC) layer dispersion and hilar- and molecular layer-ectopic DGCs are found in human and experimental temporal lobe epilepsy (TLE). Reelin is a secreted migration guidance factor important for DGC layer lamination, and its expression inversely correlates with DGC dispersion in TLE (Haas et al., J Neurosci 2002). We sought to determine whether ectopic DGCs arise from aberrantly migrating DGC progenitors in the epileptic adult dentate gyrus, and whether loss of reelin signaling underlies the migration defect. Status epilepticus (SE) was induced in adult Sprague-Dawley rats with pilocarpine. Controls received saline. Animals were given bromodeoxyuridine (BrdU) twice on day 7 and then killed 7, 9, 14 or 35 days after SE. Hippocampi from TLE patients with mesial temporal sclerosis or autopsy controls were collected. Reelin- or eGFP-transfected Cos7 cells were cultured (3 days), conditioned media concentrated, and reelin protein confirmed by immunoblot. Dentate gyrus/hippocampal explants from 7-14 day old mice were cultured in Matrigel or as organotypics for 2-4 days in serum-free media with Reelin- or eGFP-conditioned media, or reelin function-blocking CR50 antibody. Hippocampal cryosections and explants were immunostained for BrdU, Prox1, reelin and disabled-1, and neuroblast, interneuron and proliferation markers. BrdU- or Prox1-immunolabeled cells in tissue sections, and chains/dispersed cells migrating out of Matrigel explants, were quantified and compared to controls (ANOVA, post-hoc t-test). Immunofluorescence double-labeling was analyzed by confocal microscopy. Pulse-chase BrdU labeling and double-label immunofluorescence showed accelerated proliferation and ectopic migration of DGC progenitors. Immature neuronal marker expression revealed neuroblast chain migration from subgranular zone to hilus and molecular layer, leading to the accumulation of hilar- and molecular layer-ectopic, Prox1-expressing DGCs. NeuN- or Prox1-expressing ectopic DGCs, not present in controls, looked remarkably similar in epileptic rat and human dentate gyrus. Expression of reelin by dentate interneurons decreased and disabled-1 in DGC precursors increased (indicating absent reelin activation) after SE in rat. Neuroblast migration in control explants showed chain migration and few dispersed cells; CR50 antibody incubation significantly increased chain migration. Treatment with reelin-conditioned media significantly reduced chain migration and increased dispersed cells. The findings indicate that SE impairs reelin signaling in adult rat DGC progenitors, leading to aberrant chain migration and ectopic DGC formation during epileptogenesis that remarkably resembles human TLE. Moreover, reelin directly influences neuroblast migration in dentate explants, suggesting that persistent reelin expression in adult dentate gyrus serves to maintain DGC neurogenesis. (Supported by PACE, NINDS.)