Abstracts

RATIONALE: The ability to follow the development of an adult stem cell through its maturation into a functional neuron has many potential experimental applications. Dissociated cell culture lacks the extracellular matrix and support present in the intact brain while in vivo analysis of stem cells only allows end-stage examination of cell fate. In these experiments we attempted to follow neurogenesis from cell division, through migration and differentiation in slice cultures from rat or mouse brain to determine if the normal process of neurogenesis in the dentate gyrus of the hippocampus occurs in vitro.
METHODS: Organotypic hippocampal slices cultures were prepared from P6 Sprague Dawley rat brain or from P1 C57Bl6 mouse brain, according to Stoppini[scquote]s method (J. Neurosci. Methods, 1991, 37: 173-182) and grown for 14 or 28 days before being used in the experiments. Slices cultures were pulsed with 0.5 mM Bromodeoxyuridine (BrdU) and media was replaced after 24 hours. Slices were fixed at 2 weeks or 4 weeks and processed for BrdU and multiple labels immunofluorescence. Primary antibodies were chosen that recognize immature and mature astrocytes (S-100b polypeptide), immature neurons (b-tubulin, TUJ1), mature neurons (neuronal nuclear antigen clone A60, NeuN and high molecular weight neurofilament, NFH), mature astrocytes (glial fibrillary acidic protein, GFAP), mature oligodendrocytes (RIP), and immature/mature astrocytes and oligodendrocytes (adenomatous polyposis coli tumor suppresser gene, APC).
RESULTS: Confocal imaging revealed a high number BrdU labeled neural and glial progenitor cells throughout the slice. At 1 week post-BrdU, no cell that had undergone cell division expressed mature neuronal markers including NFH or NeuN. However, by 3 weeks post-BrdU pulse, NeuN labeled cells began to appear in the granule cell layer of the dentate indicating neuronal differentiation.
CONCLUSIONS: These results demonstrate that neurogenesis occurs in the dentate gyrus of organotypic hippocampal slice cultures. This in vitro model offers the ability to finely control and manipulate microenvironmental conditions critical to proliferation, differentiation and survival of neurons. The slice culture preparation may be useful in examining mechanisms of induction and manipulation of neurogenesis in models of neurological disorders such as epilepsy.
[Supported by: NIH IK24NS02128
PJH and MKM are supported by the Glaucoma Research Foundation and the Steve and Mary Kirsch Foundation.]