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Anais da Academia Brasileira de Ciências

versão impressa ISSN 0001-3765versão On-line ISSN 1678-2690

An. Acad. Bras. Ciênc. v.72 n.3 Rio de Janeiro set. 2000

http://dx.doi.org/10.1590/S0001-37652000000300031 

FORMATION OF CORTICAL TISSUE FROM SLICES MAINTAINED IN VITRO: A MODEL FOR RADIAL AND TANGENTIAL MIGRATION STUDIES*

CECILIA HEDIN-PEREIRA1, ELIZABETH C. P. DEMORAES2, MARCELO F. SANTIAGO1, ROSALIA MENDEZ-OTERO1 AND ROBERTO LENT2

1Instituto de Biofísica Carlos Chagas Filho
2Departamento de Anatomia, ICB
Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.

 

During cerebral cortex development, neurons cease to proliferate in the ventricular zone and migrate predominantly on radial glia (RG) to the surface of the cortical plate. There they uncouple from the RG and differentiate. However, recently, neurons that originate in extracortical proliferative regions were shown to migrate tangentially to the cortex giving rise to inhibitory neurons. We propose a new model to study cellular and molecular interactions in these different migratory streams based on an in vitro slice culture assay. Coronal slices (350mm) of E15 hamster brains were obtained with a tissue chopper and plated in Petriperm membranes where culture medium was added. After 2-7 days in the incubator (5%CO2) a thin layer of tissue was apparent outside the slice. Vimentin and GFAP immunoreactivity demonstrated that radial glial processes emerge from the outside of the slice border in two ways: unfasciculated, in which case the newly formed tissue emerged just outside of the slice border, or in bundles which defasciculated further out. DIC imaging showed round cells closely adhered to the glial processes. Double-labeling of GFAP and MAP-2 characterized these cells as neuronal precursors attached to glial fibers. Class III b tubulin immunoreactivity revealed differentiated neurons in a denser neuronal plexus where RG processes defasciculated. Specific markers were used to further characterize their neuronal identity and neurons were immunolabeled for MAP-2, 9-O-acetylated gangliosides and LAMP. Differentiated astrocytes were also found in the tissue. Neurons within the marginal zone (MZ) of the slice were immunolabeled for calretinin and reelin. A subpopulation of these calretinin positive neurons could be found attached to radial glia out of the slice. Many migratory profiles were found both within the MZ, seemingly migrating horizontally, and attached to the radial glial fascicles outside of the slice migrating radially. Our results show that a cortical tissue is organized from brain slice cultures based on the outward migration of neuronal precursors on radial glia. Migrating cells derived from at least two origins were identified - MZ and VZ neuronal precursors. These cells are able to migrate and differentiate several hundred microns from the slice border. MZ cells are capable of switching from a tangential migratory route into radial migration supported by glia. We propose that this model will be extremely useful to study cellular and molecular mechanisms underlying migration in the cerebral cortex. — ( June 27, 2000 )

 

* Supported by PRONEX/ MCT, FAPERJ, CNPq.
E-mail: hedin@biof.ufrj.br

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