Examination of granule layer cell count, cell density, and single-pulse brdu incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro

Abstract

Adult neurogenesis in the dentate gyrus is assuming an increasingly important role in supporting hippocampal-dependent learning and the modulation of mood and anxiety. Moreover, injury to the developing postnatal dentate gyrus has profound effects on neurogenesis and hippocampal learning throughout life. Organotypic hippocampal slice cultures represent an attractive model for studying neurogenesis both in the early postnatal and adult hippocampus, as they retain much of their anatomical and functional circuitry in vitro. Ongoing neurogenesis has been recently demonstrated in organotypic hippocampal slice cultures. However, cell proliferation, one of the critical components of neurogenesis, has yet to be characterized in this culture system. We examined single-pulse S-phase bromo-deoxyuridine (BrdU) labeling in the dentate granule layer with respect to the septotemporal position of origin of the slice culture, the medium the cultures were grown in, and the time the cultures were maintained in vitro up to 14 days, when they are believed to have matured to a near adult state. Using single 10-microm sections through a culture as our reference volume, we report significant effects of septotemporal position on the number of granule layer cells and the number of cells in S-phase, as estimated by short-survival (2 hours) BrdU studies. We report a declining rate of BrdU incorporation, evidence of significant structural changes within the granule cell layer, and differences in cell death between culture media over the first 14 days in vitro. We report caution with the use of BrdU cell density and changes in cell number to indirectly estimate proliferation.