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

Author:

Merighi AdalbertoORCID,Lossi Laura

Abstract

Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants missing the Reelin gene (reln-/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing ex vivo 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-GFPrelnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different reln genotypes and demonstrated that when the slices from reln-/- mutants were co-cultured with those from reln+/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion. Results: In images of the co-cultured slices from reln-/- mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of reln+/- animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from reln-/- mice were cultivated singularly or co-cultivated, supporting 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 for the possibility to study and manipulate ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective).

Funder

National Centre for the Replacement, Refinement & Reduction of Animals in Research

Publisher

F1000 Research Ltd

Subject

General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine

Reference41 articles.

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