Journal Article

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination version 2; peer review: 2 approved

Merighi, Adalberto, Lossi, Laura

2022

Overview

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-GFP relnF1/) 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).

• Publisher: Faculty of 1000 Ltd,F1000 Research Limited,F1000 Research Ltd
• Subject: Animals / Blood-brain barrier / Brain research / Cell Adhesion Molecules, Neuronal - genetics / Cell Adhesion Molecules, Neuronal - metabolism / Cerebellum / Coculture Techniques / eng / Ex vivo methods / Experiments / Extracellular matrix / Extracellular Matrix Proteins - genetics / Extracellular Matrix Proteins - metabolism / Fluorescence microscopy / Genetic engineering / Genotypes / Green fluorescent protein / Hybrids / Lamination / Longitudinal studies / Method / Mice / Mutants / Neurodegeneration / Neurogenesis / Neuronal migration / Phenotypes / Protein structure / Proteins / Purkinje cells / Purkinje neurons / Reelin / Reelin protein / Reelin Protein - genetics / Secreted proteins / Serine Endopeptidases - genetics / Serine Endopeptidases - metabolism