The authors thank Cristina Andr&#233;s Carbonell and members of the Animal Care facility of the Universidad de Valencia for technical assistance. We also want to thank Isabel Fari&#241;as and Sacramento R. Ferr&#243;n for reagents and sharing their equipment with us. I.M.W is funded by a Garant&#237;a Juvenil contract from the Conselleria de Educaci&#243;n de Valencia (GJIDI/2018/A/221), D.dA.D is funded by the Ministerio de Ciencia, Innovaci&#243;n y Universidades (MICINN)&#160;(FPI-PRE2018-086150). C.Gil-Sanz holds a Ram&#243;n y Cajal Grant (RYC-2015-19058) from the Spanish Ministerio de Ciencia, Innovaci&#243;n y Universidades (MICINN). This Work was funded RYC-2015-19058 and SAF2017-82880-R (MICINN).

The procedure herein described has been approved by the ethical committee in charge of experimentation, the animal welfare of the Universidad de Valencia and the Conselleria de Agricultura, Desarrollo Rural, Emergencia Clim&#225;tica y Transici&#243;n Ecol&#243;gica of the Comunidad Valenciana, and adheres to the guidelines of the International&#160;Council for Laboratory Animal Science (ICLAS) reviewed in the Real Decreto 53/2013 of the Spanish legislation as well as in the Directive 2010/63/EU of the European Parliament and of the Council.
NOTE: This protocol involves two different purposes: 1) the first study, referred to as &#8220;strategy A&#8221;, allows the analysis of the interactions between Cajal-Retzius cells (CR-cells) and early-born cortical projection neurons within the same brain hemisphere; 2) the second study, &#8220;strategy B&#8221;, is carried out in order to examine the innervation of the upper layer callosal projection neurons to the contralateral side of the neocortex.
1. Presurgery preparation
<ol>
	<li>DNA preparation
	<ol>
		<li>Transform chemically or electrocompetent E. coli DH5&#945; cells with the plasmids of interest, plate them on LB agar plates with the appropriate antibiotic, and incubate them overnight at 37 &#176;C. 
		NOTE: All plasmids used here contain the general promoter for chicken &#946;-actin (CAG) driving the expression of a fluorescent-reporter protein (CAG-mCherry and CAG-EGFP for strategy A and CAG-BFP and CAG-nEGFP-2A-mtdTomato for strategy B). All of them contain resistance to ampicillin (AMP).</li>
		<li>Pick individual colonies from each plasmid transformation and initiate a starter liquid culture in 2 mL of Luria broth + ampicillin (LB+AMP) in bacterial culture tubes during 3&#8722;4 h at 37 &#176;C with vigorous shaking (200 rpm). After, set a larger bacterial culture in a 500 mL Erlenmeyer flask adding 200 mL of LB+AMP and 1 mL of the starter culture. Incubate overnight at 37 &#176;C in the orbital shaker at 200 rpm.</li>
		<li>Use an endotoxin-free maxi-prep kit (Table of Materials) following the manufacturer&#8217;s instructions to obtain pure and concentrated plasmid DNA from the liquid cultures. Resuspend the DNA in ~50&#8722;100 &#181;L of endotoxin-free Tris-EDTA (TE) buffer to obtain concentrations of at least 5 &#181;g/&#181;L.</li>
		<li>For each surgery, prepare a solution with a final volume of 10 &#181;L containing 1 &#181;L of fast green dye, plasmid DNA solution of interest for a final concentration of 1 &#181;g/&#181;L for each plasmid, and endotoxin-free TE buffer. For example, mix 1 &#181;L of fast green dye, 2 &#181;L of a 5 &#181;g/&#181;L plasmid DNA solution, and 7 &#181;L of TE buffer.</li>
	</ol>
	</li>
	<li>Pipette pulling
	<ol>
		<li>Pull borosilicate glass capillaries (1/0.58 mm outer/inner diameter) in a vertical micropipette puller until the tip reaches a length of 1&#8722;1.5 cm and trim it using dissecting forceps at an angle of approximately 30&#176; under a dissecting scope.</li>
	</ol>
	</li>
	<li>Surgery room setup
	<ol>
		<li>Put all the equipment on the operating table (i.e., tweezers, scissors, forceps, micropipettes, needle, and needle holder). Turn on the heating pad and cover it with a sterile surgical absorbent pad. Ensure that the reservoir in the machine for inhalation anesthesia is filled with isoflurane, the oxygen tank contains enough oxygen, and the system functions properly. 
		NOTE: The surgery room and the resistant material must be kept as sterile as possible (i.e., all the material must be autoclaved before the surgery and surfaces must be sanitized with 70% ethanol). Platinum electrodes need to be carefully disinfected first with germicidal soap and second with 70% ethanol prior the surgery.</li>
		<li>Prepare 100 mL of 0.9% (w/v) sterile saline solution containing penicillin-streptomycin 1:100 and fill a 10 cm Petri dish. Place the plate on top of the heated pad to warm up the solution.</li>
		<li>Fill a 1 mL syringe with 150 &#181;L of an analgesic solution (e.g., 0.1 mg/kg buprenorphine).</li>
		<li>Load the pulled pipette with 5 &#181;L of the final plasmid DNA solution prepared in step 1.1.4. Connect the capillary to a mouth-controlled aspirator tube.</li>
	</ol>
	</li>
</ol>
2. First in utero electroporation surgery
<ol>
	<li>Place an E11.5 (strategy A) or E13.5 (strategy B) C57BL/6 pregnant mouse inside a closed induction chamber with 2.5% (v/v) isoflurane at 0.8 L/min and wait until it is anesthetized. Transfer the mouse to the heating pad and put its nose into a mask for constant delivery of isoflurane. Check for the absence of a pedal reflex as an indicator of proper anesthesia. 
	NOTE: Embryonic age is determined based on the day when the vaginal plug is observed (E0.5).</li>
	<li>Inject the pregnant female with the analgesic solution (0.1 mg/kg buprenorphine) subcutaneously. To prevent the eyes from drying during the procedure, apply one drop of eye ointment in each eye using a cotton swab.</li>
	<li>Shave the mouse abdominal area with an electric razor and wash it 2x with 70% (v/v) ethanol wipes, once with iodine wipes, and one last time with an ethanol wipe.</li>
	<li>Use scissors to make a 30 mm long incision through the skin in the right side of the animal and carefully separate the adjacent skin from the muscle with a blunt spatula. Afterwards, make a second incision in the abdominal wall.</li>
	<li>Cover the abdomen with a piece of folded tissue paper previously disinfected with 70% ethanol containing a 40 mm long slit in its center. Carefully pull the uterus out of the abdominal cavity with ring forceps. 
	NOTE: The uterus must be kept wet with the warm saline solution prepared in step 1.3.2 during the whole procedure.</li>
	<li>Preload the pulled pipettes with the DNA solution prepared in step 1.1.4. Inject ~0.5 &#181;L of the DNA solution per embryo into the lateral ventricle of the selected hemisphere using the mouth-controlled aspirator tube until the fast green dye is noticeable inside the ventricle.</li>
	<li>Place forceps-type platinum electrodes laterally around the head of the injected embryo (as shown in Figure 1A and Figure 2A). Orient the electrodes to target the desired brain region. Direct the positive pole towards the medial wall to electroporate the cortical hem (strategy A) or towards the lateral cortex (strategy B) to label cells generated in that area. 
	NOTE: In all cases, the heart and placenta must be avoided to ensure embryonic survival.</li>
	<li>Apply the specific sequence of electric pulses with a square wave electroporator following the indications shown in Table 1 (strategy A E11.5 embryos: four pulses of 25 V and 40 ms, separated by 950 ms intervals; strategy B E13.5 embryos: five pulses of 35 V and 60 ms, with 950 ms intervals).</li>
	<li>Carefully place the uterus back into the abdominal cavity with forceps, fill it with warm saline solution, and close the abdominal wall with a needle 6-0 suture. Join the two sides of the initial incision made in the skin either using a needle 6-0 suture or suture clips.</li>
	<li>Maintain the animal on the heating pad and monitor it until its recovery from anesthesia. Provide an extra dose of analgesia (150 &#181;L)&#160;in a hydrogel solution placed in its home cage.</li>
	<li>24 hours after surgery, administer an extra dose of analgesia (150 &#181;L). Continue daily monitoring by visual inspection for possible pain and distress. Observe the animal&#8217;s behavior, test its normal hind limb reflexes, and inspect the suture for possible signs of damage due to licking or scratching of the wound.&#160;&#160;</li>
</ol>
3. Second in utero electroporation
<ol>
	<li>Two days after the initial surgery, repeat steps 2.1&#8722;2.3. Although the recovery of pregnant females after the surgery is very good, check that they show normal behavior and present no signs of pain or distress before performing the second surgery (E13.5 embryos for strategy A and E15.5 for strategy B).</li>
	<li>Make a 30 mm long incision through the skin as in step 2.4 and a second incision at the abdominal wall, this time in the left side of the animal. Carefully expose the uterus on top of a disinfected tissue as described in step 2.5. 
	NOTE: Be careful not to interfere with the incision previously made in the other side.</li>
	<li>Inject around 0.5 &#181;L per embryo of the DNA solution into the lateral brain ventricle of the hemisphere previously electroporated in the case of strategy A and in the lateral brain ventricle of the contralateral hemisphere for strategy B. 
	NOTE: Use only embryos showing normal development and no signs of reabsorption.</li>
	<li>Place the electrodes around the embryo&#8217;s head as described in step 2.7, directing the positive electrode towards the lateral cortex and apply the appropriate pulses following the indications shown in Table 1 (strategy A E13.5 embryos: five pulses of 35 V and 60 ms separated by 950 ms intervals; strategy B E15.5 embryos: five pulses of 50 V and 80 ms separated by 950 ms intervals).</li>
	<li>Continue and finish the surgery as described in steps 2.8&#8722;2.10.</li>
</ol>
4. Tissue harvesting and sectioning
<ol>
	<li>Strategy A
	<ol>
		<li>Four days after the second electroporation (E17.5), perform cervical dislocation of the pregnant female and place it in a supine position.</li>
		<li>Using scissors, make a ventral incision to extract the uterine horns and with forceps place them in a Petri dish filled with 1x phosphate-buffered saline (PBS) placed on ice. 
		NOTE: The low temperature makes the anesthetization of the embryos possible.</li>
		<li>Using tweezers, extract the embryos out of the amniotic sac and transfer them with forceps to a new PBS-filled Petri dish under a dissecting scope. Carefully hold the head of the embryos using forceps and conduct brain dissection with tweezers to first remove the skin over the head and then the skull. Use a spatula to pull out the exposed brains.</li>
		<li>Collect the brains with a spoon and deposit them in a 48 well plate filled with the fixative solution (4% [w/v] paraformaldehyde [PFA] in 1x PBS). Test immediately for the successful outcome of both electroporations by examining the brains using an inverted epifluorescence microscope, for example.</li>
		<li>Fixate the embryonic brains overnight at 4 &#176;C in an orbital shaker. Wash with 1x PBS to eliminate traces of PFA. Then transfer them to PBS with antifungal preservatives (1x PBS-0.05% (w/v) sodium azide). 
		CAUTION: PFA and sodium azide are cytotoxic compounds that require special precaution during their utilization.</li>
		<li>Embed the fixated brains in 4% (w/v) low melting point agarose in 1x PBS and wait ~10 min until it solidifies. Stick them to the vibratome tissue holder using cyanoacrylate glue with the olfactory bulbs facing upwards to obtain coronal sections.</li>
		<li>Initiate the vibratome and select the desired parameters: 100 &#181;m width, 0.60 &#181;m/s speed, and 0.60 mm of amplitude.</li>
		<li>Secure the brain inside the vibratome container, fill it with 1x PBS solution, and begin collecting coronal serial sections with the help of a brush in a 48 well plate filled with 1x PBS-0.05% (w/v) sodium azide to have a complete depiction of the brain (e.g., around seven sections per well and six wells per embryo).</li>
		<li>Mount the desired sections in microscope glass slides using a fine brush. Cover them with glass coverslips. For long-term storage add mounting medium, which prevents photobleaching and photooxidation. Observe the slides under an upright epifluorescence microscope to assess electroporation efficacy.</li>
	</ol>
	</li>
	<li>Strategy B
	<ol>
		<li>Let the pups previously electroporated at E13.5 and E15.5 be born and wait until P15 to perform transcardial perfusion using the same fixative solution used in step 4.1.4.</li>
		<li>Just before perfusion, intraperitoneally administer a dose of 75/1 mg/kg ketamine/medetomidine. When the pedal reflex is lost, secure the mouse in a supine position and make a ventral incision following the middle line using scissors to expose both the rib cage and diaphragm.</li>
		<li>Cut the diaphragm and open the rib cage to gain access to the heart. Hold the rib cage using a hemostat and make an incision in the right atrium with fine scissors.</li>
		<li>Penetrate the left ventricle with a needle connected with a flexible tube to a peristaltic perfusion pump. Start transcardial perfusion, delivering at least 25 mL of 4% PFA at a constant flux of 5.5 mL/min (total time ~5 min).</li>
		<li>Dissect the brain of perfused animals. First, remove the skin over the head with scissors and forceps. Start cutting the skull using scissors and carefully pull off sections of the skull bones until they are completely removed. Finally, extract the brain with the help of a spatula.</li>
		<li>Transfer the brains to a 24 well plate and fix them with 4% PFA overnight at 4 &#176;C on an orbital shaker. Stop fixation by replacing PFA with 0.05% (w/v) sodium azide in PBS. 
		NOTE: As indicated in step 4.1.5, it is advisable to carry out an intermediate wash with 1x PBS.</li>
		<li>Repeat steps 4.1.6&#8722;4.1.9, changing the vibratome parameters for postnatal brains (40 &#181;m width, 1.20 &#181;m/s speed, and 0.5 mm of amplitude). 
		NOTE: Immunohistochemistry or immunofluorescence can be carried out to detect specific cell markers or enhance the signal of fluorescent proteins used in electroporation.</li>
	</ol>
	</li>
</ol>
5. Confocal fluorescence imaging and analysis
<ol>
	<li>Turn on the confocal microscope, place the microscope slides containing the mounted brain sections onto the microscope slides holder and select the channels at which fluorescence images will be taken (i.e., 420&#8722;460 nm for BFP, 490&#8722;540 nm for GFP, and 570&#8722;620 nm for mCherry and tdTomato).</li>
	<li>Perform sample scanning to obtain map images of each brain section at two different wavelengths for a general view of the double electroporation output. Once finished, select the 10x lens and the multi-area-Z-stack-timelapse observation mode. This will allow programming the automatic acquisition of fluorescence images at different XYZ localizations within brain sections.</li>
	<li>In each of the chosen regions (XY), set proper imaging parameters (i.e., laser intensity, photomultiplier sensitivity, and a minimum resolution of 1,024 x 1,024), as well as the depth of the scanning (Z) according to the planes of the sample where fluorescence is visible.</li>
	<li>Obtain low magnification (10x) images of all the chosen regions and export them from OIF to TIFF format using the microscope viewer software.</li>
	<li>Change to the 60x lens, repeat step 5.3 and capture high magnification images to observe the cell-cell interactions in a more detailed manner. Export them as indicated in step 5.4.</li>
	<li>Open the acquired images with any imaging software (e.g., Fiji) for further analysis.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Transcallosal+Projections+Are+Present+throughout+the+Mouse+Cortex.">Heterotopic Transcallosal Projections Are Present throughout the Mouse Cortex.</a> Frontiers in Cellular Neuroscience. 11, 36 (2017).</li><li>DeFelipe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+brain,+the+human+nature+of+cortical+circuits,+and+intellectual+creativity.">The evolution of the brain, the human nature of cortical circuits, and intellectual creativity.</a> Frontiers in Neuroanatomy. 5, 29 (2011).</li><li>Velmeshev, D., 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=Single-cell+genomics+identifies+cell+type&#8211;specific+molecular+changes+in+autism.">Single-cell genomics identifies cell type&#8211;specific molecular changes in autism.</a> Science. 364, 685-689 (2019).</li></ol>

The authors have nothing to disclose.

The study of cell-cell interactions in vivo in regions with high cellular density like the cerebral cortex is a complex task. Traditional approaches including the use of antibodies to label neurites are not suitable because of the lack of specific markers for different cell populations. The use of transgenic murine models, where a particular cell type expresses a fluorescent protein, is useful to visualize the neuronal processes, but this depends on the availability of such models. This task is even more complicated when...

The cerebral cortex is a very complex and intricately organized structure. To achieve such a degree of organization, cortical projection neurons go through complex developmental processes that require their temporal generation, migration to their final destination in the cortical plate, and the establishment of short- and long-range connections1,2. For a long time, the classical way to study corticogenesis was based on the use of knockout or knock-in murine models of genes of interest. However, this strategy, and particularly the use of conditional knockout mice, is time consuming and expensive, and sometimes presents additional problems regarding the existence of genetic redundancy or the lack of specific CRE drivers, among other issues. One of the approaches that arose to try to address those problems and that is nowadays extensively used to study cortical development is in utero electroporation3,4. In utero electroporation is a technique used for somatic transgenesis, allowing in vivo targeting of neural stem cells and their progeny. This method can be used to label cells by the expression of fluorescent proteins5,6, for gene manipulation in vivo (i.e., gain or loss of function assays)7,8,9, for isolating electroporated cortices in vitro and culturing cells8,10. Moreover, in utero electroporation permits temporal and regional control of the targeted area. This technique has numerous applications and has been widely used to study neuronal migration, stem cell division, neuronal connectivity, and other subjects8,9,11,12.
The current manuscript describes the use of an in utero electroporation variant, termed double in utero electroporation, to analyze the interactions of cells in the cerebral cortex with different temporal and spatial origins. These studies are extremely complex to complete when employing murine models because they require the combined use of several transgenic lines. Some of the applications of the protocol described in this paper include the study of close interactions among neighboring cells, as well as interactions between distant cells through long-range projections. The method requires performing two independent in utero electroporation surgeries, separated temporally and spatially, on the same embryos to target different cell populations of interest. The advantage of this approach is the possibility of manipulating gene function in one or both types of neurons using wild type animals. In addition, these functional experiments can be combined with the expression of cytoplasmic or membrane-tagged fluorescent proteins to visualize the fine morphology of targeted cells, including dendrites and axons, and analyze possible differences in cellular interactions in comparison with a control (i.e., cells only labeled with the fluorescent protein).
The protocol delineated here is focused on the study of cellular interactions inside the neocortex, but this strategy could also be used to examine interactions with extracortical areas that can be targeted using in utero electroporation, like the subpallium or the thalamus13,14, or cell-cell interactions in other structures, like the cerebellum15. Targeting of different areas is based on the orientation of the electrodes and on the ventricle where the DNA is injected (lateral, third, or fourth). With the strategy described here, we can label a substantial number of cells, which is useful to evaluate general changes in connectivity/innervation in functional experiments. Nevertheless, to study fine changes in connectivity, one can use modified versions of in utero electroporation to get sparser labeling and identify single cells16. In summary, double in utero electroporation is a versatile method that allows targeting temporally and spatially separated cell populations and studying their interactions in detail, either in control conditions or combined with functional experiments, considerably reducing temporal and economic costs.

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Braun Surgical</td><td>1050044</td><td></td></tr><tr><td>Dissecting scope</td><td>Zeiss</td><td>stemi 305</td><td></td></tr><tr><td>Dumont Forceps #5 Fine Forceps</td><td>Fine Science Tools (FST)</td><td>11254-20</td><td></td></tr><tr><td>ECM830 Square Wave Electroporator</td><td>BTX</td><td>45-0052</td><td></td></tr><tr><td>Electric Razor</td><td>Oster</td><td>76998</td><td></td></tr><tr><td>Endotoxin-free TE buffer</td><td>QIAGEN</td><td>1018499</td><td></td></tr><tr><td>Ethanol wipes</td><td>BFHCVDF</td><td></td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>Fine Science Tools (FST)</td><td>11150-10</td><td></td></tr><tr><td>Eye ointment</td><td>Alcon</td><td>682542.6</td><td></td></tr><tr><td>Fast Green dye</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools (FST)</td><td>14069-09</td><td></td></tr><tr><td>Fluorescence LEDs</td><td>CoolLED</td><td>pE-300-W</td><td></td></tr><tr><td>Genopure Plasmid Maxi Kit</td><td>Roche</td><td>3143422001</td><td></td></tr><tr><td>Halsted-Mosquito Hemostats (suture)</td><td>Fine Science Tools (FST)</td><td>91308-12</td><td></td></tr><tr><td>Heating Pad</td><td>UFESA</td><td>AL5514</td><td></td></tr><tr><td>Inverted epifluorescence microscope</td><td>Nikon</td><td>Eclipse TE2000-S</td><td></td></tr><tr><td>Iodine wipes</td><td>Lorsoul</td><td></td><td></td></tr><tr><td>Isofluorane vaporizer</td><td>Flow-Meter</td><td>A15B5001</td><td></td></tr><tr><td>Isoflurane</td><td>Karizoo</td><td>586259</td><td></td></tr><tr><td>Ketamine (Anastemine)</td><td>Fatro Ib&amp;eacute;rica SL</td><td>583889-2</td><td></td></tr><tr><td>Kimtech precision wipes</td><td>Kimberly-Clark</td><td>7252</td><td></td></tr><tr><td>LB (Lennox) Agar GEN</td><td>Labkem</td><td>AGLB-00P-500</td><td></td></tr><tr><td>LB (Lennox) broth GEN</td><td>Labkem</td><td>LBBR-00P-500</td><td></td></tr><tr><td>Low-melting point agarose</td><td>Fisher Scientific</td><td>BP165-25</td><td></td></tr><tr><td>Medetomidine (Sedator)</td><td>Dechra</td><td>573749.2</td><td></td></tr><tr><td>Microscope coverslips</td><td>Menel-Gl&amp;auml;ser</td><td>15747592</td><td></td></tr><tr><td>Microscope Slides</td><td>Labbox</td><td>SLIB-F10-050</td><td></td></tr><tr><td>Mounting medium</td><td>Electron Microscopy Sciences</td><td>17984-25</td><td></td></tr><tr><td>Mutiwell plates (24)</td><td>SPL Life Sciences</td><td>32024</td><td></td></tr><tr><td>Mutiwell plates (48)</td><td>SPL Life Sciences</td><td>32048</td><td></td></tr><tr><td>NaCl (for saline solution)</td><td>Fisher Scientific</td><td>10112640</td><td></td></tr><tr><td>Needle 25 G (BD Microlance 3)</td><td>Becton, Dickinson and Company</td><td>300600</td><td></td></tr><tr><td>Orbital incubator S150</td><td>Stuart Scientific</td><td>5133</td><td></td></tr><tr><td>P Selecta Incubator</td><td>J. P. Selecta, s.a.</td><td>0485472</td><td></td></tr><tr><td>Paraformaldehyde</td><td>PanReac AppliedChem</td><td>A3813</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma -Aldrich</td><td>P4333</td><td></td></tr><tr><td>Peristaltic perfusion pump</td><td>Cole-Parmer</td><td>EW-07522-30</td><td></td></tr><tr><td>Platinum Tweezertrode, 5 mm Diameter</td><td>Btx</td><td>45-0489</td><td></td></tr><tr><td>Reflex Skin Closure System - 7mm Clips, box of 100</td><td>AgnThos</td><td>203-1000</td><td></td></tr><tr><td>Reflex Skin Closure System - Clip Applyer, 7mm</td><td>AgnThos</td><td>204-1000</td><td></td></tr><tr><td>Ring Forceps</td><td>Fine Science Tools (FST)</td><td>11103-09</td><td></td></tr><tr><td>Sodium azide</td><td>PanReac AppliedChem</td><td>122712-1608</td><td></td></tr><tr><td>Surgical absorbent pad (steryle)</td><td>HK Surgical</td><td>PD-M</td><td></td></tr><tr><td>Suture (Surgicryl PGA 6-0)</td><td>SMI Suture Materials</td><td>BYD11071512</td><td></td></tr><tr><td>Syringe 1ml (BD plastipak)</td><td>Becton, Dickinson and Company</td><td>303172</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>Vertical Micropipette Puller</td><td>Sutter Instrument Co</td><td>P-30</td><td></td></tr><tr><td>Vertical microscope</td><td>Nikon</td><td>Eclipse Ni</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S</td><td></td></tr></tbody></table>

double in utero electroporation to target temporally and spatially separated cell populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Interactions between neighboring cells originated in distal places and at different times: Cajal-Retzius cells (CR-cells) and early migrating cortical projection neurons (strategy A)
The interaction of CR-cells and early cortical projection neurons was previously described as necessary to regulate somal translocation via nectin and cadherin adhesion molecules using a double electroporation strategy8. CR-cells originate&#160;from the neuroepithelium at t...

Watch this Scientific Journal Video about Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations at JoVE.com

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

double-utero-electroporation-to-target-temporally-spatially-separated

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.3K Views.  Universidad de Valencia. Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Video: Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

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

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

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

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

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

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

Biology

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Developmental Biology

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 ...

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 ...

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

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

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

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

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

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

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

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

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

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

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

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Chapters in this video

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

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Murine In Utero Electroporation