Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler mouse phenotype. In reln(-/-) mutants, neurons are mispositioned in layered brain areas such as the cerebellar cortex. We demonstrate that in cultured cerebellar slices, one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs). Methods: We generated mouse hybrids (L7-GFP reln F1/) with GFP-tagged PNs, directly visible under fluorescence microscopy. We cultured singularly or in combination the slices from mice with different reln genotypes and used Voronoi tessellation and geographic information systems (GIS)-based spatial statistics to validate microscopic observations. Results: In co-cultured slices from reln(-/-) mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than in slices of reln(+/-) animals, thus indicating a rearrangement of the cortical architecture toward normality. The mean roundness factor, area disorder, and roundness factor homogeneity differed when slices from reln(-/-) mice were cultivated singularly or co-cultivated with slices from reln(+/-) mice. Analysis of Central Feature, Mean Center, Median Center, Directional Distribution, Standard Distance, Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, Global Moran’s I, Anselin Local Moran’s I, and Getis-Ord G* were fully supportive of Voronoi’s results giving further insight on the role of Reelin in cerebellar development. Our approach demonstrated mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo. Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it to study and manipulate ex vivo the activity of a bioactive protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the avoidance of severe surgery (3Rs perspective).