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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			<td>Sodium azide</td>
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			<td>BYD11071512</td>
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			<td>Syringe 1ml (BD plastipak)</td>
			<td>Becton, Dickinson and Company</td>
			<td>303172</td>
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			<td>Tissue Culture Dish 100 x 20 mm</td>
			<td>Falcon</td>
			<td>353003</td>
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			<td>Vertical Micropipette Puller</td>
			<td>Sutter Instrument Co</td>
			<td>P-30</td>
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			<td>Vertical microscope</td>
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			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
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</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

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

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

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 ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.