We are grateful to Lihong Yin for technical assistance. NVD is a recipient of a NARSAD Young Investigator Award and is also supported by grants from NIH (5 K99 MH095825-02). Research in the Fishell lab is supported by the National Institute of Health, National Institute of Mental Health (5 R01 MH095147-02, 5 R01 MH071679-09), National Institute of Neurological Disorders and Stroke (5 R01 NS081297-02, 1 P01 NS074972-01A1) and the Simons Foundation.

All animals were treated in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee of the NYU School of Medicine.
Mouse Strains 
Swiss Webster female mice provided by TACONIC were used for these experiments. In order to specifically target superficial layer interneurons, e15.5 embryos were used.
Note: The plasmid used in this work (Dlx5/6.eGFP plasmid 3&#8201;&#181;g/&#181;l) was generated using standard cloning techniques. The eGFP cDNA was cloned into a Dlx5/6-Pmin-polyA plasmid. This plasmid is available upon request (demarn02@nyumc.org).
1. Preparation of Microinjection Pipettes
<ol>
	<li>Set the puller Heat #2 to 860.</li>
	<li>Place and secure the glass capillary near the filament. Check that the outer diameter (OD) of the pipette is roughly 30 &#956;m.</li>
	<li>Press the &#8220;pull&#8221; button.</li>
	<li>Carefully remove the capillary. The bottom part is the needle. Discard the top part.</li>
</ol>
2. Anesthesia Procedure
<ol>
	<li>Prepare an isoflurane containing chamber. Note: Isoflurane is the preferred anesthetic due to its fast action and low incidence of side effects. Pentobarbital is another option; however, tolerance of this drug is variable and may lead to a higher mortality rate than isoflurane.</li>
	<li>Set the flow of isoflurane to 4-5% in 0.5-1 L/min O2.</li>
	<li>Make sure that the valve for the chamber is open whereas the valve for the head adaptor is closed.</li>
	<li>Place the pregnant mouse in the chamber.</li>
	<li>Allow the mouse to go under the effect of anesthesia. This will take about 2-3 min. Check the breathing rate. If the mouse begins hyperventilating, lower the dose of isoflurane.</li>
	<li>After the mouse is anaesthetized, promptly remove it from the chamber. Place it ventral side up on the heating pad and insert the head in the mask that will continue to supply isoflurane throughout the surgery. Make sure to switch the flow of isoflurane from the chamber to the tubing connecting the head adaptor piece. Put a drop of eye lubricant in each eye and close the eyes.</li>
	<li>Set the heating pad to 36 &#176;C degrees to minimize hypothermia.</li>
	<li>Check for the absence of foot and tail reflexes by pinching the rear paws and the tail.</li>
</ol>
3. Surgery
<ol>
	<li>Cleanse the skin covering the abdomen with 70% ethanol.</li>
	<li>Use forceps to pull the skin upwards. After each surgery, wash all instruments with detergent and water, and sterilized at 72 &#176;C O/N.</li>
	<li>Make a small vertical incision (about 2 cm) along the midline starting midway between the third and fourth pairs of mammary glands. Use fine surgery scissors. Take care to not damage the abdominal wall.</li>
	<li>Gently separate the skin from the peritoneum.</li>
	<li>Visualize the arteries and veins on the abdominal wall. Make another 2 cm vertical incision through the peritoneum (avoiding the vessels) to expose the abdominal cavity.</li>
	<li>Clamp the skin and abdominal wall on each side avoiding clamping the arteries. Note: If the arteries are accidentally punctured, sacrifice the animal immediately by performing cervical dislocation or isoflurane overdose.</li>
	<li>Pre warm the sterile PBS to 37 &#176;C. Cover the clamps with PBS moist Kim wipes.</li>
	<li>Moisturize the exposed abdominal cavity with PBS. Repeat this step several times while performing the surgery.</li>
	<li>Using round forceps, gently pull the embryonic chain (e15.5) away from the cavity. Let the chain rest on the wet wipes. Start by exposing one half of the uterus first. Once the embryos in it are electroporated, bring it back inside, then work on the second half.</li>
</ol>
4. Electroporation
<ol>
	<li>Continue to use round forceps to manipulate the embryos.</li>
	<li>Visualize the lateral ventricles. The lateral ventricles run along the midline (Figure 1b).</li>
	<li>Add Fast Green (Stock: 10% w/v) to the DNA solution for a 1:20 mixture to visualize it during the injection. Prepare the DNA solution by dissolving the pellet obtained from a standard maxi-prep purification protocol in distilled water.</li>
	<li>Use pre pulled micropipettes with a plunger to inject 1 ml of DNA solution (Dlx5/6-eGFP, 3&#8201;&#181;g/&#181;l) with Fast Green. Cut the tip of the micropipette with a fine forcep #5 (Dumont) to leave 6 mm length from the beginning of the shoulder to the end of the tip of the needle before loading the pipette. Hold the head of the embryo with round tweezers. Enter the brain with the pipette at a 45&#176; angle aiming for the lateral ventricle located near the midline. If the needle penetrates through the ventricle, slowly retract the pipette as you use the plunger to inject DNA. The ventricle should turn green when the solution begins to fill it. At this point, stop moving the pipette and inject 1 ml of DNA. Gently, withdraw the pipette.</li>
	<li>Set the CUY21 electroporator to five 45V pulses of 50 msec spaced by 950 msec.</li>
	<li>Use 5 mm paddle electrodes for e15.5 embryos. Dip the electrodes in PBS to ensure efficient current transmission.</li>
	<li>Place the electrode paddles around the embryo&#8217;s head with the positive paddle on the ventricle containing the DNA solution. Slightly lower the positive paddle with respect of the negative one to create a ventral current through the ganglionic eminences. This manipulation will minimize ectopic expression in pyramidal cells.</li>
	<li>After placing the electrodes, deliver a pulse by pressing the foot pedal once.</li>
	<li>Remove the electrodes and wipe them clean. Repeat steps 4.6-4.9 with each embryo.</li>
	<li>After electroporating all the embryos, pour 3 ml of PBS into the abdominal cavity and return them to the abdominal cavity.</li>
	<li>First suture the abdominal wall with a curved needle and 5-0 silk suture and then the skin. Apply Lidocaine (4%) on the wound. Administer 120 mg/kg of Aspirin intra&#160;peritoneum for analgesia. 
	Note: The entire procedure should be performed in 20 min or less. It is advisable that beginners only electroporate a few embryos to keep the operation time short. Prolonged surgeries will decrease the survival rate of the embryos.</li>
	<li>Remove the animal from the anesthesia tubing and allow recovery on the heating pad on a paper wipe.</li>
	<li>Return animal to the cage, keep it on the heating pad at 37 &#176;C and monitor the health status. The animals generally tolerate the surgery well and begin to move slowly soon after they are removed from the anesthesia tubing. If excessive bleeding or lethargy is observed, sacrifice the animal immediately by performing IACUC approved euthanasia procedures such as cervical dislocation or anesthesia overdose.</li>
	<li>Return cage to the vivarium. The females will give birth naturally.</li>
</ol>

<ol>
	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. 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=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., 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=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. W., Wray, S., Mahon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, J., 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=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472, 351-355 (2011).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> Journal of visualized experiments : JoVE. (31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

The authors have nothing to disclose.

Limitations of the Technique
While this technique allows for cell autonomous analysis of cell processes, it is not suitable for population analysis. The electroporations are very sparse with less than a thousand cells electroporated per brain. As a consequence, the technique cannot be used to assess behavioral consequences arising from the genetic manipulation of CGE-derived interneurons.
While electroporations carried out at e13.5-e14.5 target MGE-derived subtypes,...

The bulk of cortical GABAergic interneurons originate from two transient embryonic structures named the medial and caudal ganglionic eminences (MGE and CGE respectively)4. Parvalbumin and somatostatin expressing interneurons originate in the MGE whereas Calretinin (Cr), Vasointestinal peptide (VIP) and Reelin (Re) expressing interneurons originate from the CGE. These interneuron subtypes can be distinguished by their birthdates. MGE derived subtypes are born between embryonic day 9.5 (e9.5) and e16.55,6. In contrast, CGE derived interneurons are born from e12.5 through e18.5 with their production peaking at e15.56. The genetic targeting of this late born population, however, remains elusive.
The murine distal-less (Dlx) genes are exclusively expressed in the developing ventral forebrain3. GABAergic interneurons and striatal projection neurons but not cortical pyramidal cells express Dlx1,2,5, and 6 genes at early developmental stages3. Indeed, the Dlx genes are expressed in the MGE and CGE subventricular zone (SVZ) in all GABAergic progenitors. Expression of these genes becomes restricted to select subtypes at postmitotic stages7-9. Previous experimental evidence showed that the Dlx5/6 enhancer element allows for the selective targeting of GABAergic lineages in transgenic mouse models2. We tested the use of one of these enhancer elements in the context of episomal expression in the developing mouse brain. We sub cloned the Dlx5/6 enhancer element together with a minimal promoter and the enhanced green fluorescent protein (eGFP) in a bluescript (BS) backbone plasmid (Figure 1). We introduced the plasmid by means of in utero electroporation at e15.5 to selectively target Cr-, VIP and Re- subtypes3,8,10. Our technique allows for sparse electroporation, which facilitates the reconstruction of morphological features of singe cells. In addition, the exceptionally high levels of gene expression in cortical GABAergic neurons allows for functional studies. We carried out loss and gain of function studies using several wild type and dominant negative genes11.

<table><tbody><tr><td>Electroporator&amp;nbsp; with pedal</td><td>Protech International</td><td>CUY21</td><td></td></tr><tr><td>5 mm paddle electrodes&amp;nbsp;</td><td>Protech International</td><td>CUY650P5</td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Kent Scientific</td><td>DCT-15</td><td></td></tr><tr><td>Sutter Instruments P30 Puller&amp;nbsp;</td><td>Sutter Instruments</td><td>3282322</td><td></td></tr><tr><td>Fluovac Anesthesia Systems</td><td>Harvard Apparatus</td><td>726425</td><td></td></tr><tr><td>Delicate Operating Scissors 4.75&quot; Straight Sharp/Sharp</td><td>Roboz</td><td>RS-6702</td><td></td></tr><tr><td>5-0 Silk Black Braid 18&quot; C-1 Box 36&amp;nbsp;</td><td>Roboz</td><td>SUT-1073-21</td><td></td></tr><tr><td>Micro Clip Applying Forceps 5.5&quot;</td><td>Roboz</td><td>RS-5410</td><td></td></tr><tr><td>2 Clamp scissors</td><td>Roboz</td><td>RC-4894</td><td></td></tr><tr><td>Holding forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11031-15</td><td></td></tr><tr><td>Glass capillary tubing&amp;nbsp;</td><td>FHC</td><td>27-30-0</td><td>Borosil 1.0 mm OD x 0.75 mm ID</td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258&amp;nbsp;</td><td></td></tr><tr><td>Sterile PBS</td><td>Life Technologies</td><td>20012-027</td><td></td></tr></tbody></table>

subtype-selective electroporation of cortical interneurons

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

We adapted the in utero electroporation technique to achieve cell type specific targeting of maturing neurons. To drive the expression of eGFP in CGE-derived interneurons, we used the Dlx5/6 enhancer element and restricted our injections to e15.5, the stage when the majority of CGE-derived interneurons are generated. We carried out the analysis at P8 and P15 11(Figures 1 and 2). We confirmed the ventral origin of electroporated neurons by co electroporating a...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

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

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

subtype-selective-electroporation-of-cortical-interneurons

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.8K Views.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Video: Subtype-selective Electroporation of Cortical Interneurons

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 ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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, ...

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

Developmental Biology

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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.

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 ...

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 ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Subtype-selective Electroporation of Cortical Interneurons

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Chapters in this video

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

Neonatal Pial Surface Electroporation

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

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

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

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

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

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development