Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Ex utero Electroporations with Green Fluorescent Protein Expression Plasmid

1. Plasmid DNA injection solutions are prepared with CAG-eGFP DNA ²⁷ diluted to the final working concentration of 0.33 mg/ml in ddH₂O. Qiagen Endo-Free Maxi-Preps are used to purify the plasmid from transformed bacteria. Fast green dye at ~0.02% (w/v final) is added to the DNA solution as an injection tracer.
2. To prepare the surgical area, spray down bench top and dissecting microscope stage with a 70% ethanol solution and wipe dry. Spray surgical scissors and forceps with 70% ethanol prior to dissection and wipe dry.
3. Timed Pregn....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
DMEM/F12 + GlutaMAX	GIBCO	10565
G5 Supplement 100X	Invitrogen	17503-012
B27 Serum-Free Suppl. 50X	Invitrogen	1504-044
Pen / Strep Liquid 100X	Invitrogen	15140-122
HBSS 500 ml	GIBCO	14025
Culture insert collagen coated	Costar	3492
Bovine skin gelatin	Sigma	G9382
H–chst 33342	Invitrogen	H1399
Bovine Serum Albumin	Sigma	7906
EndoFree Plasmid Maxi Kit	Qiagen	12362
Equipment
BTX 830 Electroporator	Harvard Apparatus	450052
Tweezer electrodes 10mm	Harvard Apparatus	450166
Incubator	Billups Rothenberg	MIC-101
Hamilton syringe (5uL)	Hamilton	87930
Hamilton syringe needle	Hamilton	7803-04	Specify 1" and style 4
Dumont #5 Forceps	FST	11251-10
Fine Scissors Tough Cut 9 cm	FST	14058-09