Published: April 3rd, 2013
This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.
Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.
The mammalian cerebral cortex forms through the concerted proliferation, migration and differentiation of successively generated neurons. Each neuron is born in the ventricular zone (VZ) and migrates from the VZ into the intermediate zone (IZ), forming the cortical plate (CP) 1. As they pass through different cortical domains, the migrating neurons display multiple modes of migration 2,3 that depend on the extracellular environment and other cellular elements (e.g. radial glia) within the developing tissue. Cortical neurons then arrest migration at the top of the forming cortical plate in the coincident processes of neuronal migration ar....
1. Ex utero Electroporations with Green Fluorescent Protein Expression Plasmid
The embryonic rodent cortex exhibits a transverse neurogenetic gradient throughout the developmental period, such that lateral neocortex is approximately 1 day more mature than dorsal medial neocortex 28. The bulk of layer 6 neurons are thus generated (i.e. exhibit their final S-phase) on E12 in lateral cortex (also called Field 405) and at E13 in the dorsal medial cortex (also called Field 15). Preplate splitting begins approximately 1 day after Layer 6 neuron generation and thu.......
We have improved and evaluated an experimental model - whole hemisphere explants - for the study of early cortical development (E13-E15). The model has proven useful for the analysis of migration and differentiation of the excitatory neuron lineage that constitutes layer 6 of the cerebral cortex 25,37 . The principle advantages of the system are 1) organotypic growth for 2 DIV, 2) simplicity of preparation, and 3) experimental access to the neurons for electroporation, pharmacological manipulation and imaging .......
This work was supported grants from NINDS (NS066071) and the NIAAA. (P50AA017823) to ECO. The authors thank Dr. Robert Quinn and the staff in the Department of Laboratory Animal Resources for animal care. We thank Judson Belmont for technical support, Nicole Belletier for assistance as a Summer Undergraduate Research Fellow (SURF). We also thank Dr. David Cameron for comments and edits on an earlier version of the manuscript.....
|DMEM/F12 + GlutaMAX
|G5 Supplement 100X
|B27 Serum-Free Suppl. 50X
|Pen / Strep Liquid 100X
|HBSS 500 ml
|Culture insert collagen coated
|Bovine skin gelatin
|Bovine Serum Albumin
|EndoFree Plasmid Maxi Kit
|BTX 830 Electroporator
|Tweezer electrodes 10mm
|Hamilton syringe (5 uL)
|Hamilton syringe needle
|Specify 1" and style 4
|Dumont #5 Forceps
|Fine Scissors Tough Cut 9 cm
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