Name: double in utero electroporation to target temporally and spatially separated cell populations
Uploaded: 2020-06-14T13:00:00
Description: Watch this Scientific Journal Video about Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations at JoVE.com

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 Parliamen...

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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>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9518-25G</td>
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			<td>Baby-mixter hemostat (perfusion)</td>
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			<td>Rb Pharmaceuticals Limited</td>
			<td>921425</td>
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			<td>CAG-BFP plasmid</td>
			<td>Kindly provided by U.M&#252;ller Lab</td>
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			<td>CAG-EGFP plasmid</td>
			<td>Kindly provided by U.M&#252;ller Lab</td>
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			<td>CAG-mCherry plasmid</td>
			<td>Kindly provided by U.M&#252;ller Lab</td>
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			<td>CAG-mtdTomato-2A-nGFP plasmid</td>
			<td>Kindly provided by U.M&#252;ller Lab</td>
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			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
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			<td>Cotton Swabs</td>
			<td>BFHCVDF</td>
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			<td>Cyanoacrylate glue</td>
			<td>B. Braun Surgical</td>
			<td>1050044</td>
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			<td>Dissecting scope</td>
			<td>Zeiss</td>
			<td>stemi 305</td>
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			<td>Dumont Forceps #5 Fine Forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td>11254-20</td>
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			<td>ECM830 Square Wave Electroporator</td>
			<td>BTX</td>
			<td>45-0052</td>
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			<td>Electric Razor</td>
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			<td>76998</td>
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			<td>Endotoxin-free TE buffer</td>
			<td>QIAGEN</td>
			<td>1018499</td>
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			<td>Ethanol wipes</td>
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			<td>Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td>11150-10</td>
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			<td>Eye ointment</td>
			<td>Alcon</td>
			<td>682542.6</td>
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			<td>Fast Green dye</td>
			<td>Sigma-Aldrich</td>
			<td>F7252-5G</td>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14069-09</td>
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			<td>Fluorescence LEDs</td>
			<td>CoolLED</td>
			<td>pE-300-W</td>
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			<td>Genopure Plasmid Maxi Kit</td>
			<td>Roche</td>
			<td>3143422001</td>
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			<td>Halsted-Mosquito Hemostats (suture)</td>
			<td>Fine Science Tools (FST)</td>
			<td>91308-12</td>
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			<td>Heating Pad</td>
			<td>UFESA</td>
			<td>AL5514</td>
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			<td>Inverted epifluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE2000-S</td>
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			<td>Iodine wipes</td>
			<td>Lorsoul</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Isofluorane vaporizer</td>
			<td>Flow-Meter</td>
			<td>A15B5001</td>
			<td></td>
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		<tr>
			<td>Isoflurane</td>
			<td>Karizoo</td>
			<td>586259</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Anastemine)</td>
			<td>Fatro Ib&#233;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>
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		<tr>
			<td>Low-melting point agarose</td>
			<td>Fisher Scientific</td>
			<td>BP165-25</td>
			<td></td>
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		<tr>
			<td>Medetomidine (Sedator)</td>
			<td>Dechra</td>
			<td>573749.2</td>
			<td></td>
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		<tr>
			<td>Microscope coverslips</td>
			<td>Menel-Gl&#228;ser</td>
			<td>15747592</td>
			<td></td>
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			<td>Microscope Slides</td>
			<td>Labbox</td>
			<td>SLIB-F10-050</td>
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			<td>Mounting medium</td>
			<td>Electron Microscopy Sciences</td>
			<td>17984-25</td>
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			<td>Mutiwell plates (24)</td>
			<td>SPL Life Sciences</td>
			<td>32024</td>
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			<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 in utero electroporation is a powerful method for studying the important aspects of mammalian corticogenesis such as the interaction between neuron cells, generated at different locations and developmental ages. The main advantage of this technique is the ability to examine unperturbed interactions among these different cell populations in a quick, detailed, and inexpensive manner. One striking implication of our method is that it facilitates a better understanding of how similar our wiring is altered in neuronal disorders like autism and schizophrenia.This study is not restricted to the neocortex but it can also be employed to analyze cell to cell interactions, in exocortico areas such as the sepalium or cerebellum. One of the most challenging steps is injection of DNA into the lateral ventricles of early embryos. The use of a bright light source is essential for successful injections.For each surgery first prepare a 10 microliter solution containing one microliter of fast green dye, a one microgram per microliter plasmid DNA solution of interest for each plasmid, and the appropriate volume on Endotoxin free TE buffer. And preload a glass microcapillary pipette with the appropriate prepared DNA solution. For the first in utero electroporation surgery, apply ointment to the eyes of an appropriately timed anesthetized pregnant mouse, and use a razor to shave the abdomen.Disinfect the exposed skin two times with sequential 70%ethanol and iodine wipes before proceeding with the incision on the right side of the abdomen. Once the uterus is accessible use ring forceps to carefully extract the organ. Using a mouth controlled aspirator tube, inject approximately 0.5 microliters of solution into the lateral ventricle of the left hemisphere of E11.5 embryos for strategy A.Or into the lateral ventricle of the right hemisphere of E13.5 embryos for strategy B, until the fast green dye is visible inside the ventricle.When all of the solution has been injected, place forceps type platinum electrodes laterally around of the head of the injected embryos, with the positive pole oriented to the medial wall to target the cortical hem for strategy A, or oriented to the lateral cortex for strategy B, taking care to avoid the heart and placenta. Use a square-wave electroporater to apply the appropriate sequence of electric pulses for each embryonic age, as indicated in the table. When all of the embryos have been injected and electroporated, use forceps to carefully return the uterus to the abdominal cavity, and fill the abdomen with warm saline.Using a needle 6-0 suture, close the abdominal wall, and carefully close the skin incision. Then return the animal to its home cage with monitoring until full recovery. Two days after the initial surgery, confirm that the mouse is exhibiting normal behavior, and presents no signs of pain or distress.Before preparing the animal for the second in utero electroporation as just demonstrated. Inject 0.5 microliters of the desired DNA solution into the left lateral brain ventricle of E13.5 embryos for strategy A, and into the left lateral brain ventricle of E15.5 embryos for strategy B.After each injection, place the electrodes with the positive pole oriented to the lateral cortex of the injected hemisphere for strategy A and strategy B.And electroporate embryos as indicated in the table. When the electroporation is finished, return the uterus to the abdominal cavity, and complete the surgery and post-surgical care as demonstrated.When implementing strategy A, four days after the second electroporation, harvest the uterus of the embryonic day 17.5 pregnant female mouse into a petri dish of PBS on ice. And use tweezers to transfer the embryos into a new dish of PBS under a dissecting microscope. Using forceps, carefully grasp the head of each embryo to facilitate removal of the skin over the head and skull.Use forceps or a spatula to lift out the exposed brains. Then use spoon to transfer each brain into individual wells of a 48 well plate containing around 600 microliters of fixative solution per well. When all of the brains have been collected, fix the brains at four degrees Celsius overnight on an orbital shaker, followed by three ten minute washes in PBS.After the last wash, embed the brains in 4%low melting point agarose in PBS for about ten minutes. After the agarose has solidified, use cyanoacrylate glue to secure each brain to a vibratome tissue holder olfactory bulb side up. Place the holder inside the vibratome container, and fill the container with PBS.Then use the vibratome and a brush to obtain 100 micrometer thick coronal serial sections. Transferring up to seven sections per well and six wells per embryo, in a new 48 well plate containing 600 microliters per well of fresh PBS, supplemented with anti-fungal preservatives as they are collected. When all of the sections have been acquired, use a fine brush to mount each section onto a glass microscope slide, and cover them with a glass coverslip for assessment of the electroporation efficacy on an upright epifluorescence microscope.When implementing strategy B, on postnatal day 15, after confirming a lack of response to topinch, secure the mouse in the supine position, and use scissors to make a midline skin incision to expose the ribcage and diaphragm, and cut the diaphragm to gain access to the heart. Using fine scissors, make an incision in the right atrium, and use a 25 gage needle connected to a flexible tube to a peristaltic perfusion pump to penetrate the left ventricle. When the needle is in place, deliver at least 25 milliliters of 4%paraformaldehyde by transcardial perfusion, at a constant flux at 5.5 milliliters per minute.After approximately five minutes, stop the perfusion, and use scissors to remove the skin over the head. Remove the skull, and use a spatula to remove the brain tissue. Place the brain into one well of a 24 well plate containing 1.5 milliliters of fresh 4%paraformaldehyde, for an overnight incubation at four degrees Celsius on an orbital shaker.Then acquire 40 micrometer sections of the postnatal brains as just demonstrated. For embryonic and postnatal brain slice imaging, place the slides containing the mounted brain sections of interest onto a microscope slide holder. Introduce the slider holder in the confocal microscope, and select the appropriate channel for the fluorofores used in the experiment.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, in the multi-area Z-stack time lapse observation mode. In each of the select XY regions, set the appropriate imaging perimeters as well as the depth of the scanning along the Z axis according to the planes of the sample, in which the fluorescence is visible.Obtain low magnification images of all of the chosen regions, and export the images from OIF to TIFF format in the microscope viewer software. Then select the 60x lens, and capture high magnification images as just demonstrated, to observe the cell-to-cell interactions at a more detailed level. The temporal gap between the double in utero electroporation surgeries allows CAG toresial cells targeted on embryonic day 11.5 in one of its places of origin to reach the marginal zone of the lateral neocortex including the somatosensory area, in time to establish contacts with cortical projection neurons, labeled on embryonic day 13.5.The leading processes of projection neurons expressing enhanced GFP profusely uprise in the marginal zone of the cortex, and intermingle with the processes of M-cherry expressing cataretzial cells. In utero electroporation at embryonic day 13.5, using a BFP expressing plasmid facilitates the labeling of projection neurons across different cortical layers. A second electroporation in the contralateral side of embryonic day 15.5 targets the upper layer callosal projection neurons subpopulation, but not the lower layer projection neurons that develop at earlier stages.Upper layer targeted neurons extend their axons to the contralateral hemisphere, with the characteristic arborization pattern. High magnification allows a more detailed visualization of callosal axon innervation of the targeted projection neurons within the contralateral hemisphere. When attempting this protocol, it is important to plan electroporation timing, the hemisphere to inject, and the position of the electrodes according to the cell populations to be targeted.Following this procedure all the methods, such as electroporation is the interesting one. For both cell types can be performed to study possible circuit alterations in vivo. Using this technique, it is possible to study fine interactions between two different targeted cells.Including new science information in vivo, by the electroporation of fluorescent synaptic proteins.

Research

JoVE Journal

Neuroscience

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

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 ...

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

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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 ...

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

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 ...

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

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

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

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 ...

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

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

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

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

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