Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures

Generation of dentate granule cells and its modulation by glutamate receptor antagonists, growth factors and pilocarpine-induced seizure-like activity was investigated in rat hippocampal slice cultures derived from 1-week-old rats and grown for 2 weeks. Focussing on the dentate granule cell layer facing CA1 and the immediate subgranular zone, exposure for 3 days to the NMDA receptor blocking agents MK-801 (10 μM) or APV (25 μM) in the culture medium, increased the number of TOAD-64/Ulip/CRMP-4 (TUC-4)-positive cells as counted in the slice cultures at the end of the 3-day treatment period. Exposure to IGF-I (200 ng/ml) and EGF (20 ng/ml) also increased the number of TUC-4-positive cells. Combining APV with IGF-I/EGF had an additive effect. Similar results were obtained by 3 days treatment with the AMPA receptor antagonist CNQX (25 μM). Surprisingly, addition of 5 mM pilocarpine reduced the number of TUC-4-positive cells, just as combining pilocarpine with the neurogenesis-stimulating compounds, prevented or reduced the increase of TUC-4-positive cells. None of the treatments were found to induce dentate granule cell death within the observed period. Labeling of dividing cells by adding 5-bromo-2-deoxyuridine (BrdU) to the culture medium did not result in cells double-labeled with BrdU and TUC-4. The induced increase in TUC-4-positive cells therefore represent neuronal differentiation of existing neural precursor cells when investigated at the 3-day time point. We conclude that 3 days treatment of 2-week-old hippocampal slice cultures with IGF-I and EGF and NMDA and AMPA glutamate receptor antagonists increase granule cell neurogenesis from preexisting neural precursors.