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.

1. Ex utero Electroporations with Green Fluorescent Protein Expression Plasmid
<ol>
	<li>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.</li>
	<li>To prepare the surgical area, spray down bench top and dissecting microscope stage ...

The authors declare that they have no competing financial interests.

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 ...

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 arrest and dendritogenesis 4.
Cortical development is initiated between embryonic days 11-13 (E11-13) 5 through establishment of the primordial plexiform layer 6 or preplate (PP), a layer of pioneer neurons that overlies the VZ. Prospective layer 6 cortical neurons (i.e. the first cortical neurons born in the VZ) then orient their somata in a stereotypical pattern and coalesce into a distinct layer within the PP 7. These events split the preplate into a superficial marginal (future cortical layer 1) and a deep zone, the latter composed of subplate cells (transient cortical layer 7). This process, termed preplate splitting, is a foundational event in the future growth of the cerebral cortex 8.
Many genetic mutations have been identified that disrupt various aspects of cortical development 9. Cortical development can also be negatively impacted by exposure to ingested toxins such as cocaine 10 and alcohol 11. Because cortical malformations that arise during development are likely contributors to neurological disorders (e.g. autism, schizophrenia), empirical investigations of perturbations to cortical development are inherently important. It is therefore of considerable importance to establish approaches to study cortical development that allow rapid assays of genetic or toxin effects but that also preserve the possible interactions between differentiating neurons, other cell types and extracellular matrix (ECM) during this early period of brain development 12.
Slice explants 13 have provided such a system and have been widely used to assay cortical neuron development 14-16. However, slice assays can suffer from the drawback that neuronal migration and lamination can be abnormal 17 possibly due to damage to the meningeal cells that surround the developing brain and anchor the radial glial scaffold. As radial glial fibers are an important substrate for cortical neuron migration 18 disruption of the basal lamina by slicing may locally disrupt radial glial architecture and lead to altered cortical migration. In addition the sliced surfaces of explants provides a region of dead cells that may alter the normal composition of the ECM in these areas.
More recent approaches have focused their analysis on cells located deep in the slice that are surrounded by appropriate healthy cell types and ECM. However, in some cases these newer approaches can require that the original thick cultured slice be cryo-sectioned or paraffin-sectioned after fixation so that the relatively normal interior of the slice is made available for analysis 19-21. Both the original vibratome sectioning to prepare the live slices for culture as well as the subsequent cryosectioning of fixed slices for analysis require care and effort for these assays to work.
To provide a simple, complementary approach for studies of early cortical development, we have modified an existing slice approaches 13 to facilitate studies of early cortical development. We have developed a whole hemisphere explant model similar to an existing E14 whole hemisphere model that involved shaking cultures at 65 rotations per minute and permitted organotypic growth for 16-18 hr 22,23 . In our approach, whole hemisphere explants are placed on semi-permeable membrane 13 with in a high oxygen culture atmosphere 21,24 to extend organotypic cortical growth for 48 hr. This approach also allows for consistent electroporation of developing cortical neurons. The embryos are removed from the uterus and electroporated to introduce plasmid DNA and the telencephalon is then dissected. Each hemisphere is isolated and placed medial side down on a collagen-coated filter. The explant is then cultured for a period of 48 hr, a period that encompasses preplate splitting 8. During the culture period, L6 neurons develop from precursor to differentiated neuron 25, correctly positioned within the cortical plate. Throughout this period the developing neuron is surrounded by the appropriate ECM and cell types that would confront the corresponding cell in vivo. This system has already proved valuable in deciphering the cellular events that underlie ethanol toxicity 26, layer 6 formation and preplate splitting 7,25 .

<table border="1">
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		<tr>
			<td>Reagents</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>DMEM/F12 + GlutaMAX</td>
			<td>GIBCO</td>
			<td>10565</td>
			<td>&nbsp;</td>
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			<td>G5 Supplement 100X</td>
			<td>Invitrogen</td>
			<td>17503-012</td>
			<td>&nbsp;</td>
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			<td>B27 Serum-Free Suppl. 50X</td>
			<td>Invitrogen</td>
			<td>1504-044</td>
			<td>&nbsp;</td>
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			<td>Pen / Strep Liquid 100X</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
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			<td>HBSS 500 ml</td>
			<td>GIBCO</td>
			<td>14025</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Culture insert collagen coated</td>
			<td>Costar</td>
			<td>3492</td>
			<td>&nbsp;</td>
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			<td>Bovine skin gelatin</td>
			<td>Sigma</td>
			<td>G9382</td>
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			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
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			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>7906</td>
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			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>&nbsp;</td>
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			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>BTX 830 Electroporator</td>
			<td>Harvard Apparatus</td>
			<td>450052</td>
			<td>&nbsp;</td>
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			<td>Tweezer electrodes 10mm</td>
			<td>Harvard Apparatus</td>
			<td>450166</td>
			<td>&nbsp;</td>
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			<td>Incubator</td>
			<td>Billups Rothenberg</td>
			<td>MIC-101</td>
			<td>&nbsp;</td>
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			<td>Hamilton syringe (5 uL)</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td>&nbsp;</td>
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			<td>Hamilton syringe needle</td>
			<td>Hamilton</td>
			<td>7803-04</td>
			<td>Specify 1" and style 4</td>
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			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>11251-10</td>
			<td>&nbsp;</td>
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			<td>Fine Scissors Tough Cut 9 cm</td>
			<td>FST</td>
			<td>14058-09</td>
			<td>&nbsp;</td>
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ex utero electroporation and whole hemisphere explants: a simple experimental method for studies of 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 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...

Watch this Scientific Journal Video about Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development at JoVE.com

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

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.

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

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

ex-utero-electroporation-whole-hemisphere-explants-simple

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JoVE Journal

Neuroscience

12.8K Görüntüleme.  SUNY Upstate Medical University. Bu protokol içerir geliştirilmiş bir eksplant yordam açıklanır Ex utero Embriyonik fare genelinde hemisferlerin elektroporasyon, diseksiyon ve kültür. Hazırlanması erken kortikal gelişim sırasında gen fonksiyonu farmakolojik çalışmalar ve testler kolaylaştırır.