We thank Dr. Shirin Bonni for providing the pSil-GFP construct, Dr. Alper Uzun for the illustration of Figure 1, and the Leduc Bioimaging facility for confocal microscopy. EMM is supported by the Career Award for Medical Science from the Burroughs Wellcome Fund, a NARSAD Young Investigators Award, and NIH NCRR COBRE P20 RR018728-01. SBL is supported by NIH NCRR COBRE P20 RR018728-01, and has received support from PHS NRSA 5T32MH019118-20.

1. Preparing Culture Solutions and Media (not in video)
<ol>
 <li>Prepare 1 liter of Complete Hank's balanced salt solution (HBSS) containing 1x HBSS, 2.5 mM Hepes (pH7.4), 30 mM D-glucose, 1 mM CaCl2, 1 mM MgSO4 and 4 mM NaHCO3. Add double distilled water (ddH2O). Filter sterilize with a 0.2-&mu;m filter and store at 4 &deg;C.</li>
 <li>Prepare slice culture medium using 35 mL of Basal Medium Eagle media, 12.9 mL of Complete HBSS, 20 mM D-glucose (1.35 mL of 1 M solution), 1 mM Glutamax (0.25 mL of a 200 mM solution), 0.5 ml of Penicillin-streptomycin 100x stock. Filter sterilize with a 0.2-&mu;m filter, then add heat-inactivated horse serum to a final concentration of 5%.</li>
 <li>Prepare Laminin working solution by making a 1 mg/mL Laminin stock solution with sterile distilled deionized water. Prepare 100 &mu;L aliquots in 0.5 mL eppendorf tubes and freeze at -80 &deg;C.</li>
 <li>Prepare Poly-L-lysine working solution by adding 5 mL of sterile H2O to 5 mg of poly-L-lysine to make a 1 mg/mL stock solution. Prepare 1 mL aliquots and freeze at -20 &deg;C.</li>
 <li>Prepare coating solution by diluting 1 mL of poly-L-lysine and 100 &mu;L of laminin to a final volume of 12 mL with sterile water. Make this solution fresh each time.</li>
</ol>
2. Preparing Organotypic Slice Inserts (not in video)
<ol>
 <li>Prepare two six-well plates with one culture insert per well using sterile forceps. Add 2 mL of sterile ddH2O underneath the culture inserts.</li>
 <li>Add 1 mL of the coating solution on top of the membrane taking care not to puncture the membrane. Incubate overnight in a humidified incubator at 37 &deg;C and 5% CO2.</li>
 <li>Remove coating media and wash membrane with sterile H20 three times. Let inserts dry before use. Add 1.8 mL of slice culture medium and place in 37 &deg;C incubator. Wrap the unused plates with Parafilm and store at 4 &deg;C for up to 4 weeks.</li>
</ol>

3. Preparing for Electroporation (not in video)
<ol>
 <li>Prepare RNAi constructs for electroporation. Double strand RNA hairpin inserts were cloned into a pSilencer vector. The plasmid contains: 1) a U6 promoter that drives the double strand RNA generation; and 2) a GFP expression cassette driven by the CMV promoter8,9. This plasmid has been previously described by Konishi and colleagues9 and others7,8. Plasmids are purified using a Qiagen maxi-prep kit, and used at a concentration of 1 mg/mL.</li>
 <li>In order to visualize DNA while injecting it, prepare a 0.5% fast green dye solution and use it at 1:20 with the DNA to be injected (usually 20 &mu;L of DNA with 1 &mu;L of fast green). Left over DNA-fast green dye mixture can be stored at -20 &deg;C for up to a week.</li>
 <li>Clean dissection area, dissecting tools and vibratome vessel with 70% ethanol. Chill complete HBSS so that it is ice cold. Chill vibratome vessel by packing ice around it inside the vibratome with some water for rapid cooling. Prepare 3% low melting point agarose using complete HBSS. Microwave for 1 min. Avoid over boiling. Keep in a 42 &deg;C waterbath until use.</li>
 <li>Electroporation parameters are set as follows. For an E15 embryo use 35 V, 5 pulses, 100 ms length, 900 ms interval between pulses. For older animals, to ensure electroporation, use higher voltage up to 50 V or increase the number of pulses up to 8 pulses. To prevent damaging the tissue in younger animals, use either fewer pulses or up to 2 pulses and lower voltage up to 25 V. These parameters may be varied and determined empirically depending on the age of the animal.</li>
</ol>
4. Dissection and Electroporation (in video)
<ol>
 <li>After euthanizing a pregnant female, dissect embryos out into ice cold complete HBSS. Keep each embryo in their individual placental sacs.</li>
 <li>Dissect embryo out and cut off the head after the first vertebra. Keep in ice cold complete HBSS.</li>
 <li>For injection, place the head on a piece of Parafilm on top of a petri dish. Using custom made Hamilton syringe (see Table I) inject about 6 to 8 &mu;L of DNA:fast green dye mix through the third ventricle in order to fill in both lateral ventricles in the cortical vesicles. Alternatively, injection can be done directly into each lateral ventricle.</li>
 <li>For the ex vivo electroporation, use BTX-tweezer platinum electrodes. Place the positive electrode towards the side of the cortex you want to electroporate i.e. top of the head for dorsal cortex.</li>
 <li>After electroporation, incubate the heads on ice for at least 5 min before dissecting.</li>
 <li>Dissect brains in ice cold HBSSby making a small incision on the side of the head and peeling the skin off the sides of the head. Next, with fine forceps gently peel away the pia from the brain. Remove the intact brain from the skull, taking care not to damage the cortex.</li>
</ol>
5. Embedding and Sectioning of Electroporated Cortices (in video)
<ol>
 <li>Transfer 3% low melting point agarose into a large mold placed on ice. The bottom of the mold will start to solidify faster which will prevent brains from sinking down to the bottom of the mold. Gently, transfer brains with fine forceps one by one after removing excess buffer with a Kimwipe or filter paper. Use a 10 &mu;L pipet tip to swirl the brains inside the mold to ensure maximum interface between the agarose and brain tissue.</li>
 <li>Orient the brains to ensure all brains are in the same orientation and at about the same level in the agarose. Let the agarose solidify for about 5 min. Use bonding adhesive (crazy glue) to attach the agarose blocks so that the olfactory bulbs are standing up. Once the blocks are attached, immediately add ice cold HBSS and trim the agarose to make sure individual slices are obtained for each brain.</li>
 <li>To slice the blocks, set the vibratome's velocity to a low speed (about half the maximum) and set the blade vibration frequency at the highest setting. Generate 250-&mu;m thick coronal slices. Retrieve slices using a bent fine spatula and transfer them to tissue wells with a fine brush or forceps.</li>
 <li>In tissue culture hood, transfer slices into coated inserts. Add 500 &mu;L of slice culture media to each insert to make the transfer easy. Up to 5 slices can be placed per insert. Remove excess media from the top of the slices and incubate at 37 &deg;C in humidified incubator.</li>
</ol>
6. Culture and Analysis of Organotypic Slices (in video)
<ol>
 <li>In order to maintain healthy slices, fresh media should be added at least every other day underneath the membrane by replacing half of the media each time.</li>
 <li>In order to analyze slices after the desired days in culture, fix slices in the membrane. Wash with 1x phosphate saline buffer (PBS) at 37 &deg;C three times for 10 min each time. Next, fix with 4% Paraformaldehyde (PFA) overnight at 4 &deg;C or for 1 hr at room temperature.</li>
 <li>Slices can be analyzed with different cellular markers or stained with Hoechst alone to visualize electroporated and non-electroporated cells. Permeabilize and block slices for 2 hr at room temperature with 10% goat serum, 0.1% triton in 1x PBS with gentle shaking.</li>
 <li>Stain with Hoechst for 1 hr at room temperature, wash 3 times with 1x PBS 10 min each time with gentle shaking.</li>
 <li>To mount slices cut the membrane with a scalpel, and use a fine forceps to transfer the membrane containing slices to a frosted glass slides in a water chamber. Up to 5 slices can be placed per glass slide. Remove excess water, and add a drop of Flourmount solution to each brain slice. Gently place a coverslip on top of the brain slices and remove any air bubbles. Analyze slices using a confocal microscope.</li>
</ol>
7. Alternative Paraffin Embedding of Organotypic Slices (not in video)
<ol>
 <li>Organotypic slices can also be embedded for paraffin for a finer morphological analysis, to this end the membrane containing the organotypic slices can be fixed in 4% PFA as described above.</li>
 <li>The fixed slices are embedded in 1% agarose (pre-warmed to 37 &deg;C), and solidified in ice for 30 min. The agarose blocks can then be post-fixed in 4% PFA at 4 &deg;C for 30 min.</li>
 <li>The agarose block containing the organotypic slice will then be embedded in paraffin and processed for immunofluorescence as previously described10.</li>
</ol>
8. Representative Results
A diagrammatic representation of the electroporation of murine cortex and culture of organotypic slices is shown in Figure 1. This method is a useful strategy for rapid assessment of the function of genes involved in neuronal development11. Depending on the amount of DNA electroporated and the embryonic stage at electroporation, the transfection efficiency will vary. Slices will start expressing GFP at least 8 hours post-electroporation and cells will undergo the normal sequence of neurogenic events (proliferation, migration, and early neuronal differentiation) in culture. Figure 2 shows an electroporated brain slice that is expressing a control pSilencer- GFP vector and one can observe neuronal progenitors, migrating neurons and differentiated neurons in the slice. Organotypic slices will keep their morphology as long as they are maintained in a good media-air interface on the membranes and can be used up to at least 5 days in culture.
<img alt="Figure 1" src="/files/ftp_upload/3621/3621fig1.jpg" /> 
Figure 1. Illustration of ex vivo electroporation and organotypic slice culture assay. E14.5 embryos are dissected out, and individually injected with DNA mixed with fast green dye in order to visualize the injection site. DNA can be injected in both the lateral ventricles as depicted in the illustration or in the third ventricle in order to fill the lateral ventricles. After injection, brains are electroporated with a square wave electroporator, placing the positive electrode on the desired side of the brain. Brains are embedded in 3% low melting point agarose and sectioned using a vibratome. 250 &mu;m brain slices are placed on 0.4 &mu;m inserts and cultured up to a week. GFP can be observed after 8 hours post transfection.
<img alt="Figure 2" src="/files/ftp_upload/3621/3621fig2.jpg" /> 
Figure 2. Analysis of electroporated brain slices. Electroporated brain slices were stained for Hoescht. Dorsal cortex shows electroporated neuronal progenitors at the ventricular zone (vz). Neurons in the cortical plate (cp) are delimited by the marginal zone (mz). White arrows show migrating neurons. In this case, brains were injected at E15.5 and electroporated with a pSilencer GFP control vector. The sections represent cortical explants 4 days after electroporation. Scale bar 100 &mu;m.
Troubleshooting:
<ol>
 <li>Low transfection efficiency: Adjust the concentration of DNA used to at least 1 &mu;g/&mu;L. Always use very clean DNA from a maxi prep if necessary use an endo-free Quiagen kit to purify the DNA.</li>
 <li>Cells transfected in a different brain area than the one desired: Ensure that the electrodes are positioned correctly with the positive electrode position towards the side of the brain to be electroporated.</li>
 <li>Organotypic slices lose morphology: Change media every day and ensure the slices are not floating in media</li>
 <li>Slices come off the agarose as they are being cut in the vibratome: Ensure that a good interface is made when embedding the brains in the low melting point agarose.</li>
</ol>

<ol>
	<li>Angevine, J. B., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+study+of+cell+migration+during+histogenesis+of+cerebral+cortex+in+the+mouse.">Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.</a> Nature. 192, 766-766 (1961).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-392 (2004).</li><li>Barnes, A. P., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+axon-dendrite+polarity+in+developing+neurons.">Establishment of axon-dendrite polarity in developing neurons.</a> Annu. Rev. Neurosci. 32, 347-347 (2009).</li><li>Haydar, T. F., Bambrick, L. L., Krueger, B. K., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+for+analysis+of+proliferation,+cell+death,+and+migration+in+the+embryonic+neocortex.">Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex.</a> Brain Res. Brain Res. Protoc. 4, 425-425 (1999).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. 2002, pl9-pl9 (2002).</li><li>Guerrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-BAR+domain+of+srGAP2+induces+membrane+protrusions+required+for+neuronal+migration+and+morphogenesis.">The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.</a> Cell. 138, 990-990 (2009).</li><li>Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+regulation+of+axonal+morphogenesis+by+the+Cdh1-APC+target+SnoN.">Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.</a> Neuron. 50, 389-389 (2006).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PLoS Genet. 4, e1000111-e1000111 (2008).</li><li>Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303, 1026-1026 (2004).</li><li>Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+paraffin-embedding+of+mouse+tissues+for+GFP+visualization.">Fixation and paraffin-embedding of mouse tissues for GFP visualization.</a> Cold Spring Harb Protoc. 2009, 5298-5298 (2009).</li><li>Hand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-45 (2005).</li><li>Taniguchi, Y., Young-Pearse, T., Sawa, A., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Electroporation+as+a+Tool+for+Genetic+Manipulation+in+Vivo+to+Study+Psychiatric+Disorders:+From+Genes+to+Circuits+and+Behaviors.">In Utero Electroporation as a Tool for Genetic Manipulation in Vivo to Study Psychiatric Disorders: From Genes to Circuits and Behaviors.</a> Neuroscientist. 18, 169-179 (2012).</li></ol>

We have nothing to disclose.

These methods involving the ex vivo electroporation of plasmids encoding double stranded RNA hairpins 8 and culture of organotypic slices4 provide several distinct advantages. First, these methods allow for a rapid assessment of RNAi-derived phenotypes. The inclusion of an expression cassette encoding GFP in the same pSilencer vector that contains the U6 promoter that drives the double stranded RNA hairpin allows for a rapid identification and characterization of cells electroporated with t...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hamilton syringe </td>
 <td>HAMILTON </td>
 <td>80008</td>
 <td>31 gauge, 0.5 inches long,PT-4 (level of point beveling), 10&mu;l volume</td>
 </tr>
 <tr>
 <td>Platinum tweezertrodes</td>
 <td>BTX</td>
 <td>45-0489</td>
 <td> 5mm size</td>
 </tr>
 <tr>
 <td>ECM830 electroporator</td>
 <td>BTX</td>
 <td>45-0002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BTX Footswitch</td>
 <td>BTX</td>
 <td>45-0208</td>
 <td>For use with ECM830 electroporator</td>
 </tr>
 <tr>
 <td>Vibrating blade microtome</td>
 <td>LEICA</td>
 <td>VT1000 S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6- well dish to use with inserts</td>
 <td>FALCON</td>
 <td>353502 </td>
 <td>Contains notches to fit inserts</td>
 </tr>
 <tr>
 <td>Tissue culture inserts</td>
 <td>FALCON</td>
 <td>353090 </td>
 <td>0.4 micrometer </td>
 </tr>
 <tr>
 <td>Fast Green</td>
 <td>SIGMA</td>
 <td>F7252</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low Melting Agarose</td>
 <td>FISHER</td>
 <td>BP165-25</td>
 <td>DNA grade</td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Sigma-Aldrich</td>
 <td>L2020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma-Aldrich</td>
 <td>P5899</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Basal Medium Eagle</td>
 <td>Sigma Aldrich</td>
 <td>B-1522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HBSS 10x without Ca and Mg</td>
 <td>GIBCO</td>
 <td>14180-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES-free acid</td>
 <td>Sigma- Aldrich</td>
 <td>H4034</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

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.

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 ...

methods-for-study-neuronal-morphogenesis-ex-vivo-rnai-electroporation

Research

JoVE Journal

Neuroscience

11.7K Views.  Brown University. 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.

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

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 ...