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 with a 70% ethanol solution and wipe dry. Spray surgical scissors and forceps with 70% ethanol prior to dissection and wipe dry.</li>
	<li>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).</li>
	<li>Carefully dissect each embryo from the surrounding extraembryonic membranes and keep in ice-cold HBSS.</li>
	<li>Transfer each embryo individually to a second 10 cm Petri dish containing cold HBSS. Use a Hamilton syringe to inject 2-3 &mu;l 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.</li>
	<li>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.</li>
</ol>
2. Whole Hemisphere Explant Preparation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &deg;C tissue culture incubator for the remainder of the culture period, 1-2 DIV.</li>
</ol>
3. Fixation of Explant Tissue for Histology
<ol>
	<li>In a fume hood prepare the Pagano fixation solution by first solubilizing 8 g of paraformaldehyde in 100 ml of preheated (~80 &deg;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.</li>
	<li>To fix the explants warm the Pagano fix solution to 37 &deg;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.</li>
	<li>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 &deg;C until embedding.</li>
</ol>
4. Embedding and Sectioning for Histology
<ol>
	<li>Prepare a 10% calf skin gelatin solution by solubilizing 10 g of calf skin gelatin in 100 ml of warm water (55-60 &deg;C). Place gelatin solution on a hot plate set to 60 &deg;C and swirl periodically until solubilized.</li>
	<li>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 &deg;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.</li>
	<li>Explants in gelatin blocks are positioned olfactory bulb up and sectioned in the coronal plane at 100 &mu;m thickness. The sections are collected and stored in PBS + 0.1% sodium azide at 4 &deg;C until immunohistochemical processing.</li>
	<li>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 &mu;g/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.</li>
</ol>

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><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 + GlutaMAX</td><td>GIBCO</td><td>10565</td><td></td></tr><tr><td>G5 Supplement 100X</td><td>Invitrogen</td><td>17503-012</td><td></td></tr><tr><td>B27 Serum-Free Suppl. 50X</td><td>Invitrogen</td><td>1504-044</td><td></td></tr><tr><td>Pen / Strep Liquid 100X</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>HBSS 500 ml</td><td>GIBCO</td><td>14025</td><td></td></tr><tr><td>Culture insert collagen coated </td><td>Costar</td><td>3492</td><td></td></tr><tr><td>Bovine skin gelatin </td><td>Sigma</td><td>G9382</td><td></td></tr><tr><td>H&amp;#8211;chst 33342</td><td>Invitrogen</td><td>H1399</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>7906</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BTX 830 Electroporator</td><td>Harvard Apparatus</td><td>450052</td><td></td></tr><tr><td>Tweezer electrodes 10mm</td><td>Harvard Apparatus</td><td>450166</td><td></td></tr><tr><td>Incubator</td><td>Billups Rothenberg</td><td>MIC-101</td><td></td></tr><tr><td>Hamilton syringe (5uL)</td><td>Hamilton</td><td>87930</td><td></td></tr><tr><td>Hamilton syringe needle</td><td>Hamilton</td><td>7803-04</td><td>Specify 1&quot; and style 4</td></tr><tr><td>Dumont #5 Forceps</td><td>FST</td><td>11251-10</td><td></td></tr><tr><td>Fine Scissors Tough Cut 9 cm</td><td>FST</td><td>14058-09</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.8K Views.  SUNY Upstate Medical University. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Developmental Biology

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biology

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

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

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Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons