Analysis of Mouse Brain Sections by Live-cell Time-lapse Confocal Microscopy

Abstract

The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.

Zfra Overrides WWOX in Suppressing the Progression of Neurodegeneration

“…Many genes in common fragile sites are unstable and tend to undergo deletions and alterations [ 97 ]. WWOX, DAB1, and many proteins are in involved in neuronal migration and lamination in the developing cerebral cortex [ 97 , 98 , 99 , 100 ].…”

Advanced Techniques Using In Vivo Electroporation to Study the Molecular Mechanisms of Cerebral Development Disorders

“…A significantly diluted Cre expression vector plasmid combined with the Cre/lox system allows for nearly clonal labeling by IUE [ 96 ]. We have employed this sparse labeling technique with brain slice methodologies [ 97 , 98 , 99 ] to conduct live imaging analyses of neural stem cell behavior [ 19 , 100 , 101 ]. Live imaging of the neural cells in brain slices after IUE has also been utilized in various other contexts, such as the analysis of neuronal migration [ 9 , 102 ], and extended to the subcellular level, such as the study of microtubule dynamics using EB3 [ 103 ], the Golgi apparatus [ 104 ], and visualization of the apical junctional components and centrosomes [ 59 ].…”