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 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 .

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 Pregnant Swiss/Webster dams are sacrificed on E13 by transfer into a chamber filled with CO₂ from a pressurized cylinder and the dam is monitored until all motion has stopped for at least 1 min. After sacrifice by CO₂ inhalation the embryos are removed from the uterus and placed in a 10 cm Petri dish containing ice cold Hanks balanced saline solution (HBSS).
4. Carefully dissect each embryo from the surrounding extraembryonic membranes and keep in ice-cold HBSS.
5. Transfer each embryo individually to a second 10 cm Petri dish containing cold HBSS. Use a Hamilton syringe to inject 2-3 μl of the DNA fast green mixture into the ventricle of the left cerebral hemisphere, taking care to inject in a cortical area spatially separate from the cortical area for future analysis.
6. To electroporate, hold the embryo gently with forceps and lightly place the positive paddle of the tweezer electrode on the dorsal midline of the head and lightly place the negative paddle underneath the embryo chin. Electroporations are achieved with a BTX830 electroporator programmed to deliver five 30 V pulses of 50 msec duration, separated by 950 msec intervals. These settings are for the BTX830 model. Other systems can be used according to manufacturer's instructions.

2. Whole Hemisphere Explant Preparation

1. After electroporation the embryos are placed back into ice-cold HBSS. The brain is removed using two #5 jeweler forceps to remove the skin and cartilaginous skull from the embryo head. A forcep is then slid underneath the brain to remove the intact brain from the skull. The electroporated hemisphere (left) is then dissected away from the brain and attached mid-brain tissue is removed. Throughout the dissection procedure care is taken to not damage the meninges overlying the left cortical hemisphere.
2. Once dissected each embryo is transferred in HBSS using a pipettor with a cut 1 ml pipette tip. The hemisphere is gently expelled onto a collagen coated culture insert. Up to six hemisphere explants are arranged medial side down on each 24 mm insert. Once arranged, excess HBSS is removed from the insert and the insert is then placed into one 35 mm well of a 6 well dish. The well contains exactly 2.7 ml of media: DMEM-F12 media containing Glutamax and supplemented with 2% B-27, 1% G5 and 1% Penicillin-Strep. A few drops of the media from the well can be pipetted on top of each explant, but do not add media in excess of the original 2.7 ml per well.
3. Once the explants have been placed onto Collagen filters the 6 well dish containing the explants is placed into a Billups Rothenberg (BR) chamber (that also contains a humidifying dish of water). A 95%/5% Oxygen/CO₂ gas mixture is infused into the chamber for at least 1 min before sealing the chamber shut and disconnecting the 95%/5% Oxygen/CO₂ gas supply tube. The BR chamber is then placed into a 37 °C tissue culture incubator for the remainder of the culture period, 1-2 DIV.

3. Fixation of Explant Tissue for Histology

1. In a fume hood prepare the Pagano fixation solution by first solubilizing 8 g of paraformaldehyde in 100 ml of preheated (~80 °C) ddH₂O. Add 1-3 drops concentrated NaOH to promote solubilization and gently stir. Once solubilized mix the 8% paraformaldehyde solution 1:1 with a solution that is 500 mM sucrose, 100 mM Hepes, pH 7.4, 50 mM MgCl₂, 5 mM KCl for a final concentration of 4% paraformaldehyde in 250 mM sucrose 50 mM Hepes, 25 mM MgCl₂, 2.5 mM KCl, pH 7.4.
2. To fix the explants warm the Pagano fix solution to 37 °C. Quickly remove most of the DMEM/F12 media from each culture well and add 5 ml of Pagano Fix to each well of explants. Make sure to cover each explant completely in fix. Fix for 1 hr at RT. Do not remove explants from filter prior to 1 hr of fixation.
3. After fixation remove the Pagano fix solution and replace with Pagano solution without fix (250 mM sucrose 50 mM Hepes, 25 mM MgCl₂, 2.5 mM KCl, pH 7.4). Add a few drops of 10% sodium azide and store at 4 °C until embedding.

4. Embedding and Sectioning for Histology

1. Prepare a 10% calf skin gelatin solution by solubilizing 10 g of calf skin gelatin in 100 ml of warm water (55-60 °C). Place gelatin solution on a hot plate set to 60 °C and swirl periodically until solubilized.
2. Pour approximately 10 ml of the 10% gelatin solution into the bottom of a 10 cm Petri dish and allow 30 min to solidify. This will form a "pad" for embedding the explants. These pads can be prepared days in advance and stored at 4 °C. Use a ruler to draw a grid on the bottom of the Petri dish and transfer individual explants into each box of the grid. Remove residual Pagano solution and using a pipettor, cover each explant with warm 10% gelatin solution and allow to solidify. Resume slow addition of gelatin solution until each explant is completely surrounded by gelatin, taking care not to melt the pad by the addition of too much warm gelatin at once. Allow for the gelatin to harden for ~1 hr. Once hardened the individual explants can be cut out in small blocks of gelatin. The blocks are then post-fixed in Pagano fix for 24-48 hr prior to sectioning with a vibratome.
3. Explants in gelatin blocks are positioned olfactory bulb up and sectioned in the coronal plane at 100 μm thickness. The sections are collected and stored in PBS + 0.1% sodium azide at 4 °C until immunohistochemical processing.
4. Immunohistochemical detection is performed by transferring sections into a 24 well plate (2-3 sections per well). First block non-specific antibody binding by adding 0.5 ml PBST+B (PBS + 0.5% Triton X 100 and 2% BSA) to each well, incubate 1 hr with gentle shaking. The appropriate primary antibody is then diluted in PBST+B and 0.4 ml per well is added for overnight incubation at RT. The primary is washed out with 3X PBS washes for at least 10 min each. The secondary antibody and 2 μg/ml Hoechst 33342 nuclear stain are then diluted in PBST+B and the samples are incubated for 2 hr at RT with gentle shaking. Three PBS washes (10 min each) are performed and then the sections are mounted on slides using a 90% glycerol 50 mM Tris, pH7.4, mounting media.

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