We thank S. Fouquet and the imaging and animal core facilities of Institut de la Vision and Institut des Neurosciences de Montpellier (MRI and RAM) for technical assistance. This work was supported by fellowships from R&#233;gion Ile-de-France and Fondation ARC pour la Recherche sur le Cancer to S.C, and from Universit&#233; Paris-Saclay (Initiatives Doctorales Interdisciplinaires) to L.A., by funding from European Research Council (ERC-SG 336331, PI J. Valette) to E.H., by Agence Nationale de la Recherche under contracts ANR-10-LABX-65 (LabEx LifeSenses), ANR-11-EQPX-0029 (Equipex Morphoscope2), ANR-10-INBS-04 (France BioImaging), by Fondation pour la Recherche M&#233;dicale (Ref. DBI20141231328), by the European Research Council (ERC-CoG 649117, PI J. Livet) and by ATIP-Avenir program (PI K. Loulier).

All animal procedures described here were carried out in accordance with institutional guidelines. Animal protocols have been approved by the Charles Darwin animal experimentation ethical board (CEEACD/N&#176;5).
1. Preparation of endotoxin-free plasmids for MAGIC Markers in utero electroporation
<ol>
	<li>Bacterial transformation
	<ol>
		<li>On ice, thaw DH5 alpha competent cells stored at -70 &#176;C.</li>
		<li>Warm up the agar plates containing the appropriate antibiotic (100 &#181;g/mL ampicillin or 50 &#181;g/mL kanamycin) at 37 &#176;C.</li>
		<li>Add 1 &#181;L of 5&#8211;50 ng of MAGIC Markers plasmid DNA in 10 &#181;L of thawed DH5 alpha competent cells and incubate on ice for 10 min without mixing.</li>
		<li>For the heat shock transformation, place the aliquot in a 42 &#176;C water bath for 45 s, then place immediately on ice and wait 3&#8211;5 min.</li>
		<li>Under sterile conditions, add 230 &#181;L of SOC medium and incubate for 1 h at 37 &#176;C.</li>
		<li>Spread the content of the aliquot over the agar plate and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Plasmid culture
	<ol>
		<li>The following morning, under sterile conditions, pick up a colony from the agar plate and put it in a 14 mL tube containing 2 mL of LB medium with appropriate antibiotic. Let it incubate for the day at 37 &#176;C in a shaking incubator at 300 rpm.</li>
		<li>At the end of the day, seed 300 mL of LB with antibiotic using the 2 mL starter culture from step 1.2.1 and incubate overnight at 37 &#176;C in a shaking incubator at 300 rpm.</li>
	</ol>
	</li>
	<li>Plasmid DNA preparation
	<ol>
		<li>The following morning proceed with the purification of MAGIC Markers plasmid DNA (i.e., PB-CAG-Cytbow and Tol2-CAG-Nucbow as well as CAG-driven plasmids expressing PB and Tol2 transposases and Cre recombinase) using an endotoxin-free maxiprep kit following the manufacturer&#8217;s protocol.</li>
		<li>Elute the DNA in 200 &#181;L of sterile water and estimate its concentration using a spectrophotometer prior to storage at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation for MAGIC Markers in utero electroporation (MM IUE)
<ol>
	<li>Solution preparation
	<ol>
		<li>Warm 30 mL of 0.9% saline solution at 37 &#176;C in a water bath and keep it warm during the entire duration of the surgery.</li>
		<li>Prepare a plasmid mix containing PB-CAG-Cytbow and Tol2-CAG-Nucbow (final concentration, 0.8 &#181;g/&#181;L each), PB and Tol2 transposases (final concentration, 0.4 &#181;g/&#181;L each), CAG-seCre (final concentration, 0.16 &#181;g/&#181;L), and 0.01% Fast Green dye in PBS without Ca2+ and Mg2+. 
		NOTE: For astrocyte anatomical reconstruction purposes, the Tol2-CAG-Nucbow construct may be omitted. However, this plasmid is useful to distinguish doublets of closely juxtaposed astrocytes and when using MAGIC Markers to probe clonal relationships among astrocytes14.</li>
		<li>Prepare anesthetic solution containing 100 &#181;L of ketamine (100 mg/mL) and 100 &#181;L of xylazine (20 mg/mL) diluted in 2 mL of saline solution.</li>
		<li>Prepare analgesic solution by diluting 0.3 mg/mL buprenorphine stock solution 1:10 in saline solution.</li>
	</ol>
	</li>
	<li>Preparation of the surgery material
	<ol>
		<li>Sterilize the surgical tools (see Table of Materials) at high temperature in a glass bead sterilizer or equivalent.</li>
		<li>Place a drop of electrode gel in a 35 mm dish.</li>
		<li>Insert the micropipette in the microinjector holder, break the tip of the micropipette, and aspire the DNA solution.</li>
		<li>Place a 1 &#181;L drop of Fast Green solution (0.01%) in the lid of a 3 cm dish and, using it as a reference, adjust the diameter of the micropipette&#8217;s tip by breaking its tip with fine forceps. Adjust the pressure parameter so that equivalent size drops are produced by the microinjector, thereby enabling delivery of approximately 1 &#181;L of DNA solution per injection.</li>
	</ol>
	</li>
	<li>Preparation of the pregnant female mouse
	<ol>
		<li>Weigh the RjOrl:SWISS pregnant mouse.</li>
		<li>Perform an intraperitoneal injection with 12.5 &#181;L per gram of body weight (BW) of anesthetic solution. Wait for 5 min and verify that the mouse is asleep by pinching its toe.</li>
		<li>Once the animal is unresponsive to pinch, inject it subcutaneously with 1.6 &#181;L/g BW of analgesic solution.</li>
		<li>Add a drop of ocular gel on each eye to prevent drying during the surgery and place the animal belly up on the warming pad.</li>
		<li>Gently shave its abdomen, clean it with a pad soaked with iodine, and sanitize the shaved area with an alcohol pad.</li>
		<li>Arrange an operating field by placing sterile compresses around the shaved, cleaned, and sanitized area.</li>
	</ol>
	</li>
</ol>
3. In utero electroporation (IUE)
<ol>
	<li>Intraventricular injection
	<ol>
		<li>Cut a 2 cm-vertical incision along the midline starting in the lower part of the abdomen, through the skin, and then through the underlying muscle. Expose the uterine horns by gently manipulating the embryonic bags and assess the location of the cervix and the number of embryonic bags on each side of the cervix.</li>
		<li>Orient the brain of the E15.5 embryo to be electroporated in order to see the bregma,&#160;easily recognized as its location matches with the junction of the three main blood vessels that run along the cerebral fissures.</li>
		<li>Imagine a virtual line between the bregma (visible through the skull) and the eye; introduce the micropipette between the virtual line and the longitudinal fissure, then press the injector&#8217;s foot pedal to deliver 1 &#181;L of DNA solution in the lateral ventricle of the targeted hemisphere. 
		NOTE: When injected at the proper location, the filled lateral ventricle appears blue, indicating that it has been filled with the DNA solution.</li>
	</ol>
	</li>
	<li>Electroporation
	<ol>
		<li>Apply the electrodes (see Table of Materials), previously dipped in electrode gel, on both sides of the injected embryo with the anode covering the injected hemisphere. Press the foot pedal of the electroporator to deliver a series of four 50 ms pulses of 35 V, each separated by a 950 ms interval.</li>
		<li>Dampen the embryo with warmed saline solution. 
		NOTE: The embryos should be kept humid using warmed saline solution during the entire surgical operation and the uterus should not dry out.</li>
		<li>Repeat steps 3.1.2&#8211;3.2.2 for each embryo.</li>
		<li>Once all targeted embryos have been electroporated, replace the uterine horns in the abdomen by gently pushing them with forceps back to their original position. Fill the abdominal cavity with warmed saline solution to prevent the uterus from drying while sutures are made.</li>
		<li>First close the abdominal muscle with a continuous absorbable suture, and then the skin with multiple individual stitches (~10) using 4-0 suture.</li>
		<li>Put the animal in a clean cage, lying on its side on a clean paper towel and turn the animal to the other side every 5&#8211;10 min until it wakes up and starts moving on its own.</li>
		<li>Assess the state of the animal the following day, especially if a nest has been made. 
		NOTE: No postsurgery treatment is required. In the absence of a nest, any sign of pain (e.g., prostration, shaggy fur), and/or heavy bleeding, the animal should be euthanized without delay.</li>
	</ol>
	</li>
</ol>
4. Tissue harvesting and sectioning
<ol>
	<li>Tissue collection
	<ol>
		<li>Inject phenobarbital (100 mg/kg BW) intraperitoneally for terminal anesthesia at the desired harvesting time. In this case, the harvesting times were at postnatal (P) days P4, P7, and P21.</li>
		<li>Perform intracardiac perfusion using cold premade paraformaldehyde-based solution.</li>
		<li>Dissect out the brain and place it overnight at 4 &#176;C in the paraformaldehyde-based solution for postfixation.</li>
	</ol>
	</li>
	<li>Histology
	<ol>
		<li>The following morning rinse the brain 3x for 10 min with 1x PBS.</li>
		<li>Embed the brain in 3% agarose dissolved in 1x PBS.</li>
		<li>Cut 80 &#181;m sections using a vibrating-blade microtome.</li>
		<li>Collect sections in a 24 well plate prefilled with 1x PBS.</li>
		<li>Mount sections in the mounting medium (see Table of Materials) between the slide and coverslip. Keep the mounted slides at -20 &#176;C for optimal fluorescent protein preservation and long-term storage.</li>
	</ol>
	</li>
</ol>
5. Multichannel confocal imaging
<ol>
	<li>Microscope settings
	<ol>
		<li>Set up the configuration of the confocal microscope to separately excite mCerulean/mTurquoise2, EYFP, tdTomato/mCherry using 440, 515, and 559 nm laser lines, respectively.</li>
		<li>Use a 20x 0.8 NA (or higher NA) objective and adjust XY sampling and Z-step size according to Nyquist criteria. 
		NOTE: Images acquired with higher resolution (e.g., 60x 1.4 NA oil objective) and deconvolution algorithms will enable reconstruction of the finer details of astrocyte arbors. However, the experimenter should keep in mind that the finest astrocyte processes may not be resolved by conventional optical imaging.</li>
	</ol>
	</li>
	<li>Image acquisition
	<ol>
		<li>Find the brightest astrocytes in the electroporated area.</li>
		<li>Because labeled cells located close to the surface might appear brighter than those deeper in the slice, adjust the acquisition settings on the surface cells to avoid saturating the images.</li>
		<li>Adjust acquisition settings separately for each of the three channels while making sure to avoid pixel saturation using HiLo LUT, which displays zero values as blue and maximum pixel values as red. 
		NOTE: An adequately balanced image should display only a few blue and almost no red pixels.</li>
		<li>Acquire tiled 1,024 x 1,024 pixel Z-stacks using the motorized stage of the microscope, with a 10% overlap between adjacent stacks to subsequently enable mosaic reconstructions of the electroporated area or the zone of interest.</li>
	</ol>
	</li>
</ol>
6. Astrocyte territorial volume segmentation
NOTE: This is performed using a commercial software program (e.g., IMARIS).
<ol>
	<li>Preparation of the dataset
	<ol>
		<li>Crop the labeled astrocytes within the 3D reconstruction (250 x 250 pixels) by selecting cells entirely enclosed in the imaged tissue section. 
		NOTE: It is also possible to work directly on larger volumes if the computer used can handle them.</li>
		<li>Click on the Blue Surface button in the Object toolbar of the Surpass view to create a new Surface for each astrocyte.</li>
		<li>To obtain better visualization contrast, first adjust the minimal and maximal color contrast values by clicking on Edit | Show display Adjustment option and dragging both handles. If needed, change the channel color by clicking directly on the channel name in the Display Adjustment window in order to select a new color. 
		NOTE: Preferentially always work with the same color display to prevent visual bias.</li>
		<li>Click on Edit | Image Properties to manually indicate the voxel size in micrometers. If the voxel size is not accurate, the volume calculations will be incorrect.</li>
		<li>Save the new settings.</li>
	</ol>
	</li>
	<li>Surface segmentation
	<ol>
		<li>Use the Blue Icon to introduce a surface. A Surface icon and box will appear on the Scene list. A Volume box allows to see/hide the dataset.</li>
		<li>Select the surface line and click on Skip Automatic Creation | Edit Manually.</li>
		<li>Click on Contour | Visibility and click None. Then move to the Select view and click on the Draw button.</li>
		<li>Click on Mode to select the drawing mode.</li>
		<li>Use the fast and efficient Isoline semiautomated drawing tool. Define the cell outline by moving the mouse pointer on it. If the isoline preview does not match the astrocyte outline, adjust the mouse pointer position. To validate the preview, left click on the selection. To correct potential mistakes, use the Board window to Delete the current Z-plane contour or press Ctrl + Z.</li>
		<li>Using Slice | Position to navigate through the Z-planes, move to the next plane by changing the Z-plane number and start a new contour. Preferentially start from the middle of the cell and then move to the extremities.</li>
		<li>When contours have been drawn in all the Z-planes containing the astrocyte, click on Create Surface. Before creating a surface, check that all the components are connected. If not, select the largest or connect relevant ones before exporting volume data.</li>
		<li>Check the quantitative data displayed in the Data Panel and export statistics in spreadsheet format, saving volume value and unit.</li>
		<li>Save the surface under a new name to access it later.</li>
	</ol>
	</li>
</ol>
7. Tracing astrocyte arborization
NOTE: This is done using open access software program Vaa3D.
<ol>
	<li>Preparation of the dataset
	<ol>
		<li>Load the image stack and search for isolated astrocytes or nearby astrocytes displaying distinct colors. 
		NOTE: The resolution of the reconstruction can be improved by increasing the objective NA and sampling. Ultimately, however, due to the resolution limit of confocal microscopy, the finest astrocyte processes cannot be accurately traced. Care should be taken to avoid oversegmenting beyond the resolution of the acquired images.</li>
		<li>Using Fiji, crop 250 x 250 pixel image stacks around each astrocyte.</li>
		<li>As Vaa3D tracing is performed on only one color, select one channel and deactivate the other channels.</li>
		<li>Convert the image to RGB and save it in .tiff format.</li>
	</ol>
	</li>
	<li>Arbor tracing
	<ol>
		<li>Open the RGB image in Vaa3D. Go to Image | Data | Geometry then image resampling, and adjust X/Y and Z voxel size values. 
		NOTE: While reconstructing astrocyte arbors, care should be taken not to trace details finer than the resolution provided by the images.</li>
		<li>Open a 3D window by clicking on Visualize | 3D viewer for the entire image. Right-click on a 3D image and choose 1-right-click to define marker. Point with the cursor the center of the astrocyte and then right-click to set a marker. 
		NOTE: Click on Escape to have access to the 3D view again.</li>
		<li>Go to Advanced | 3D tracing | Vaa3D-Neuron2-auto-tracing.</li>
		<li>On the newly opened window, select the channel on which to apply the plug-in.</li>
		<li>Choose a background threshold (often between 30 and 90). Adapt this value for each astrocyte if needed.</li>
		<li>Uncheck Radius from 2D and keep Auto-down sample and auto-resample checked.</li>
		<li>Set cnn_type at 3, length_thresh at 1, and SR_ratio at 0.1.</li>
		<li>After clicking ok, ensure that the plug-in automatically generates two files based on the original name. The suffix &#8211;ini.swc and -coordinateX&#8211;coordinateY-coordinateZ-app2.swc will be added. Manually add a distinctive label to these name files in order not to overwrite them while continuing to run the plug-in.</li>
		<li>Open Object Manager, go to Neuron | Line structure, select the traced astrocyte, click on Display Mode, and select Always line mode.</li>
		<li>Go back to the 3D window, where a skeleton of the astrocyte appears. 
		NOTE: If segmentation and signal do not match, repeat steps 7.2.3&#8211;7.2.10 and adjust the threshold until they do. Make sure to re-enter the other parameters as they are reset when the plug-in is restarted.</li>
		<li>Right-click on skeleton and select first choice neuron | line#1&#8230;APP2_Tracing to access quantitative measurements that will appear on a new window Surface| Object Annotation. 
		NOTE: Due to the limited resolution of light microscopy, measurements displayed under the listed items Number of branches and Number of bifurcations provide only a rough estimate of astrocyte arbor complexity.</li>
	</ol>
	</li>
</ol>

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H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nature Biotechnology. 28 (4), 348-353 (2010).</li><li>Peng, H., Bria, A., Zhou, Z., Iannello, G., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensible+visualization+and+analysis+for+multidimensional+images+using+Vaa3D.">Extensible visualization and analysis for multidimensional images using Vaa3D.</a> Nature Protocols. 9 (1), 193-208 (2014).</li><li>Peng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+finger+boosts+three-dimensional+imaging+and+microsurgery+as+well+as+terabyte+volume+image+visualization+and+analysis.">Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis.</a> Nature Communications. 5 (1), 4342 (2014).</li><li>Loulier, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Cell+and+Lineage+Tracking+with+Combinatorial+Labels.">Multiplex Cell and Lineage Tracking with Combinatorial Labels.</a> Neuron. 81 (3), 505-520 (2014).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108 (2), 193-199 (1991).</li><li>Abdeladim, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+multiscale+brain+imaging+with+chromatic+multiphoton+serial+microscopy.">Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.</a> Nature Communications. 10 (1), 1662 (2019).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> Journal of Neuroscience Methods. 207 (2), 172-180 (2012).</li><li>Siddiqi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+Mapping+by+PiggyBac+Transposase+Reveals+That+Neocortical+GLAST++Progenitors+Generate+More+Astrocytes+Than+Nestin++Progenitors+in+Rat+Neocortex.">Fate Mapping by PiggyBac Transposase Reveals That Neocortical GLAST+ Progenitors Generate More Astrocytes Than Nestin+ Progenitors in Rat Neocortex.</a> Cerebral Cortex. 24 (2), 508-520 (2014).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes to Cells. 15 (5), 501-502 (2010).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+Improved+Tools+for+In+Utero+Electroporation+Studies+of+Developing+Cerebral+Cortex.">New and Improved Tools for In Utero Electroporation Studies of Developing Cerebral Cortex.</a> Cerebral Cortex. 19, 120-125 (2009).</li><li>Pacary, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+Genetic+Manipulation+of+Dendrites+and+Spines+in+the+Mouse+Cerebral+Cortex+and+Hippocampus+using+in+utero+Electroporation.">Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using in utero Electroporation.</a> Journal of Visualized Experiments. (65), e4163 (2012).</li></ol>

The authors have nothing to disclose.

In utero electroporation (IUE) of MAGIC Markers in cortical progenitors (Figure 1) enabled labeling of astrocytes throughout the postnatal cerebral cortex at different postnatal stages (P4-P7-P21, Figure 2). Interestingly, the stage of IUE is not critical, as electroporation performed from E13.5 to E15.5 yields similar labeling patterns concerning cortical astrocytes14. However, the location of labeled pyramidal neurons in the cortical pa...

Astrocytes play numerous vital functions in brain development and physiology1. Beside their role at the blood-brain barrier where they regulate nutrient uptake and blood flow, they actively contribute to synapse formation and function while producing neuromodulators that can alter neuronal activity and behavior2. Furthermore, astrocyte dysfunction contributes to a variety of neurological disorders3. Astrocytes located in the cerebral cortex display an elaborate morphology enabling extensive contact with neuronal processes. These contacts, essential for circuit function, also control astrocyte morphogenesis and synaptogenesis through cell-adhesion proteins4. Neuroscientists need convenient and robust tools to investigate astrocyte development and morphogenesis in their neurological models of interest. However, due to the close apposition of astrocytes&#160;to their&#160;neighbors and their uniform three-dimensional tiling, it is challenging to single out cortical astrocytes and comprehensively assess their morphology using immunomarkers.
Currently, two main genetic engineering strategies enable labeling and individualization of cortical astrocytes in situ: sparse reporter activation in transgenic mouse lines or somatic transgenesis using electroporation of reporter plasmids. The first strategy relies on breeding a floxed reporter mouse line with mice expressing an inducible form of Cre recombinase activated specifically in astrocytes upon tamoxifen delivery (e.g., Aldh1l1-CreERT25). Several disadvantages are associated with this strategy. First, breeding transgenic mice requires a large number of animals and multiple assays are typically needed to determine the proper dose of tamoxifen to provide adequately sparse labeling of cortical astrocytes. Analyzing cortical astrocyte phenotypes in a genetic mouse model of interest will require even more breeding and mouse consumption. Furthermore, in utero tamoxifen injection is known to interfere with parturition, making this strategy difficult to apply to the study of the earliest stages of astrocyte development. In vivo DNA electroporation is an alternative tamoxifen-free strategy that relies on a minimum number of animals6. Performed either at embryonic or postnatal stages, this approach consists of injecting reporter plasmids in the lateral ventricles of rodents followed by electric pulses that create pores in the cell membrane, hence allowing DNA to enter progenitor cells lining the ventricle. Subsequently, the reporter transgenes carried by the electroporated plasmids are processed by the targeted cell machinery and expressed7. Two electroporation methods have been previously described to label mouse cortical astrocytes: 1) Postnatal Astrocyte Labeling by Electroporation (PALE), which relies on the electroporation of 1&#8211;2 single-color episomal reporter plasmids at early postnatal stages4; 2) The StarTrack strategy based on in utero electroporation (IUE) of multiple single-color integrative reporter plasmids8,9,10. Although these two techniques efficiently label PrA in the cerebral cortex, they also present some limitations. In their initial version, both methods rely on a glial fibrillary acidic protein (GFAP) promoter to drive expression in astrocytes, which may bias the labelling toward radial glia as well as pial and reactive astrocytes that express GFAP more strongly than normal resting PrA11,12. Regarding PALE, other disadvantages are the late stage of electroporation, which prevents labeling of early-born PrA (or those originating from early delaminating progenitors) and analysis of early stages of astroglia development, and the use of episomal vectors that become diluted through successive divisions during the massive proliferation that PrA undergo during the first postnatal week13,14. In contrast to PALE, StarTrack is based on the embryonic electroporation of integrative reporter plasmids that allow tracking the contribution of both embryonic and postnatal progenitors to PrA. An updated StarTrack scheme relying on the ubiquitin C promoter (UbC-StarTrack) achieves broader expression of fluorescent reporters in both the neuronal and glial descent (astrocytes included) of neural progenitors15,16,17. However, in its current version, implementation of this approach is complex, as it relies on an equimolar mixture of 12 distinct plasmids expressing six fluorescent proteins (FP) with partial excitation and emission spectra overlap.
Presented here is a straightforward in utero electroporation-based multicolor labeling method using integrative reporter constructs driven by a strong and broadly active promoter to single out cortical astrocytes14. In addition, an easy image analysis pipeline using both licensed (e.g., Imaris) and open access (Vaa3D18,19,20) image analysis software is provided to segment astrocyte territorial volume and arborization, respectively. Compared to the previously described methods, this strategy relies solely on 1&#8211;2 multicolor integrative transgenes Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM21) directed to the cytoplasmic and (optionally) nuclear cell compartment whose expression is driven by a synthetic CAG promoter comprised of a cytomegalovirus enhancer, chicken &#946;-actin promoter, and rabbit &#946;-globin splice acceptor site22. This enables labeling and tracking of cortical astrocytes, from embryonic to late postnatal stages, independent of GFAP expression14,23. Each of these transgenes bears the following four distinct FP: eBFP, mTurquoise2/mCerulean, EYFP, and tdTomato/mCherry, which display minimal spectral overlap that can be easily circumvented with 1) Sequential channel acquisition; 2) Optimized excitation power and collection gain; and 3) Specific dichroic filters to collect narrow FP emission windows. The MM strategy uses Cre/lox recombination with a self-excisable Cre recombinase (seCre) to drive stochastic expression of FP in a cellular population. A single copy of MM transgene expresses FP in a mutually exclusive manner, while multiple transgenes give rise to FP combinations, creating dozens of distinct hues. Genomic integration of the transgenes is driven by the piggyBac (PB) or Tol2 transposition system24,25,26. Therefore, through in utero electroporation, the MM toolkit and the multicolor &#8216;mosaic&#8217; that it generates enable simultaneous marking of multiple adjacent cortical progenitors and the tracking of their glial descent, including cortical astrocytes, over long periods. Color contrasts resulting from the expression of distinct FP permit delineation of the contour of PrA and subsequently extract key information about their territorial volume (using IMARIS) and complex morphology (using Vaa3D). The multicolor strategy presented in detail here is a convenient and robust method that gives quick and easy access to the cortical astrocyte surface and morphology in wild type mice at various developmental stages, and is easily adaptable to investigate astrocyte anatomical features in mouse models of neurological diseases without using transgenic reporter lines.

<table>
	<tbody>
		<tr>
			<td>1.1 Bacteria transformation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Euromedex</td>
			<td>EU0400-C</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5 alpha competent cells</td>
			<td>Fisher Scientitic</td>
			<td>11563117</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice box</td>
			<td>Dutscher</td>
			<td>139959</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>60615</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Fisher Scientitic</td>
			<td>11563117</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile petri dish- 10 cm</td>
			<td>Thermo Fisher</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>462-0556H</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 Plasmid culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml culture tube</td>
			<td>Dutscher</td>
			<td>187262</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass erlenmeyer- 2L</td>
			<td>Fisher Scientitic</td>
			<td>11383454</td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td>Sigma</td>
			<td>L3522</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 Plasmid DNA preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi Plus EF</td>
			<td>Macherey-Nagel</td>
			<td>740426.10</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1 Preparation of the solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26 G x 1/2 needle</td>
			<td>Terumo</td>
			<td>8AN2613R1</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2 needle</td>
			<td>Terumo</td>
			<td>8AN3013R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma Aldrich</td>
			<td>F7272</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use polypropylene syringe, 1 mL</td>
			<td>Dutscher</td>
			<td>50002</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.2 Preparation of the surgery material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps - DeBakey Pattern- 12.5 cm</td>
			<td>FST</td>
			<td>11617-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Arched tip Forceps- 10 cm</td>
			<td>FST</td>
			<td>11071-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bead sterilizer Steri 250</td>
			<td>Sigma</td>
			<td>Z378569</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micropipette 1 mm diameter</td>
			<td>FHC</td>
			<td>10-10-L</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps - Titanium 1 mm Tips Slight Curve- 10 cm</td>
			<td>FST</td>
			<td>11651-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps - Titanium 1 mm Tips Straight- 10 cm</td>
			<td>FST</td>
			<td>11650-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight- 9 cm</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory tape</td>
			<td>Fisher Scientitic</td>
			<td>11730454</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>INJECT+MATIC</td>
			<td>No catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder - 12 cm</td>
			<td>FST</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>VWR</td>
			<td>631-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic-coated white paper</td>
			<td>Distrimed</td>
			<td>700103</td>
			<td></td>
		</tr>
		<tr>
			<td>Signagel electrode gel</td>
			<td>Free-Med</td>
			<td>15-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dish- 35 mm</td>
			<td>Dutscher</td>
			<td>056714</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer, glass dry bead, Steri 250</td>
			<td>Sigma</td>
			<td>Z378569</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.3 Preparation of the pregnant female mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol pad</td>
			<td>Alcomed</td>
			<td>1731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprecare</td>
			<td>Axience</td>
			<td>0.3 mg/ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Compress</td>
			<td>tRAFFIN</td>
			<td>70189</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td>Imalgene 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular gel</td>
			<td>tvm lab</td>
			<td>Ocry-gel</td>
			<td></td>
		</tr>
		<tr>
			<td>RjOrl:SWISS mice</td>
			<td>Janvier Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vetadine, 10% solution</td>
			<td>Vetoquinol</td>
			<td>4337400113B</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming pad</td>
			<td>Harvard Apparatus</td>
			<td>72-0493</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>Rompun 2%</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable suture Size 4-0 45 cm Suture 1-Needle 19 mm Length 3/8 Circle Reverse</td>
			<td>Novosyn</td>
			<td>C0068220</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporateur Sonidel</td>
			<td>Sonidel</td>
			<td>NEPA 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile transfer pipets (individually wrapped)</td>
			<td>Dutscher</td>
			<td>043202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers with 3 mm platinium disk electrodes</td>
			<td>Sonidel</td>
			<td>CUY650P3</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4.1 Tissue harvesting and sectioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigenfix</td>
			<td>Microm Microtech</td>
			<td>U/P0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Dutscher</td>
			<td>100266</td>
			<td></td>
		</tr>
		<tr>
			<td>Dolethal</td>
			<td>Vetoquinol</td>
			<td>DOL202</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (10X), no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>11540486</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>EMS</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Dutscher</td>
			<td>100001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vectorlabs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5. Multichannel confocal imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X oil NA 0.85</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM880</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apochromat 20x/0.8 M27</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6. Astrocyte territorial volume segmentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS 8.3 and later versions</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7. Astrocyte arborization tracing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Visualization-Assisted Analysis software suite (Vaa3D)</td>
			<td>HHMI - Janelia Research Campus /Allen Institute for Brain Science</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero electroporation of multiaddressable genome-integrating color (magic) markers to individualize cortical mouse astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

Electroporation of MAGIC Markers in embryonic cortical progenitors allows for the labeling of astrocytes from early to late stages of cerebral cortex development (Figure 1). These astrocytes were found in all cortical layers at various postnatal stages (P4, P7, P21) as they dispersed widely into the entire cerebral cortex. They were assessed with tiled confocal images acquired with a 20x 0.8 NA (or higher NA) objective and assembled as Z-stack reconstructions (Figure 2</...

Watch this Scientific Journal Video about In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes at JoVE.com

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

in-utero-electroporation-multiaddressable-genome-integrating-color

Research

JoVE Journal

Neuroscience

5.3K Views.  Institut des Neurosciences de Montpellier. Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Video: In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

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 ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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.

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.

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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, ...