An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain
Summary
This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.
Abstract
Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.
Protocol
Discussion
Neuronal migration in the RMS is an essential component of postnatal neurogenesis in the olfactory bulbs 1. Migration through the RMS occurs in a plane tangential to the surface of the brain. Tangentially migrating neuroblasts are distinct from radially migrating cells based on the location of their progenitor source, as well as the divergent fate of their final neuronal products 1, 2, 3. The relatively pure population of tangentially migrating cells in the postnatal RMS makes this anatomically de…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Dan McWhorter for narrating the protocol in the video. This work is supported by NIH Grant 5R01NS062182, a grant from American Federation for Aging Research, and institutional funds awarded to HTG.
Referências
- Ghashghaei, H. T., Lai, C., Anton, E. S. Neuronal migration in the adult brain: are we there yet. Nat. Rev. Neurosci. 8, 141-151 (2007).
- Valiente, M., Marin, O. Neuronal migration mechanisms in development and disease. Curr. Opin. Neurobiol. 20, 68-78 (2010).
- Rakic, P. Evolution of the neocortex: a perspective from developmental biology. Nat. Rev. Neurosci. 10, 724-735 (2009).
- Jaglin, X. H., Chelly, J. Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects. Trends Genet. 25, 555-566 (2009).
- Carro, M. S. The transcriptional network for mesenchymal transformation of brain tumours. Nature. 463, 318-325 (2010).
- Wu, W. Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature. 400, 331-336 (1999).
- Hu, H., Tomasiewicz, H., Magnuson, T., Rutishauser, U., U, . The role of polysialic acid in migration of olfactory bulb interneuron precursors in the subventricular zone. Neuron. 16, 735-743 (1996).
- Shapiro, E. M., Gonzalez-Perez, O., Garcia-Verdugo, M. a. n. u. e. l., Alvarez-Buylla, J., &, A., Koretsky, A. P. Magnetic resonance imaging of the migration of neuronal precursors generated in the adult rodent brain. Neuroimage. , (2006).
- Vreys, R. MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain: validation of various MPIO labeling strategies. Neuroimage. 49, 2094-2103 (2010).
- Davenne, M., Custody, C., Charneau, P., Lledo, P. M. In vivo imaging of migrating neurons in the mammalian forebrain. Chem. Senses. 30, 115-116 (2005).
- Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H., Alvarez-Buylla, A. The subventricular zone en-face: wholemount staining and ependymal. J. Vis. Exp. , (2010).
- Shen, Q. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell. 3, 289-300 (2008).
- Tavazoie, M. A specialized vascular niche for adult neural stem cells. Cell Stem Cell. 3, 279-288 (2008).
- Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. Neural Stem Cells Confer Unique Pinwheel Architecture to the Ventricular Surface in Neurogenic Regions of the Adult Brain. Cell Stem Cell. 3, 265-278 (2008).
- Polleux, F. &. a. m. p. ;. a. m. p., Ghosh, A. The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal. Sci. STKE. , L9-L9 (2002).
- Murase, S. &. a. m. p. ;. a. m. p., Horwitz, A. F. Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream. J. Neurosci. 22, 3568-3579 (2002).
- Suzuki, S. O. &. a. m. p. ;. a. m. p., Goldman, J. E. Multiple cell populations in the early postnatal subventricular zone take distinct migratory pathways: a dynamic study of glial and neuronal progenitor migration. J. Neurosci. 23, 4240-4250 (2003).
- Ghashghaei, H. T. The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone. Proc. Natl. Acad. Sci. U. S. A. 103, 1930-1935 (2006).
- Khodosevich, K., Seeburg, P. H., Monyer, H. Major signaling pathways in migrating neuroblasts. Front Mol. Neurosci. 2, 7-7 (2009).
- Jacquet, B. V. Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors. Gene Ther. 16, 1021-1033 (2009).
