Characterization of Cortical Neuronal and Glial Alterations during Culture of Organotypic Whole Brain Slices from Neonatal and Mature Mice
Staal, JA and Alexander, SR and Liu, Y and Dickson, TC and Vickers, JC, Characterization of Cortical Neuronal and Glial Alterations during Culture of Organotypic Whole Brain Slices from Neonatal and Mature Mice, PLoS One , 6, (7) Article e22040. ISSN 1932-6203 (2011) [Refereed Article]
Background: Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures
and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and
morphological alterations associated with hippocampal brain slice cultures has been well established, however, the
neuronal response of mouse cortical neurons to culture is not well documented.
Methods: In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in
cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults
(P50+). Cultures were prepared using the membrane interface method.
Results: Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlueTM (cell respiration)
analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more
mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a
decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic
brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial
organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression
(p,0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular
protrusions from slice edges, including cells that expressed both glial and neuronal markers.
Conclusion: In summary, we present a substantial evaluation of the viability and morphological changes that occur in the
neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.