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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Electroporator&#160; with pedal</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY21</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mm paddle electrodes&#160;</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad&#160;</td>
			<td style="width:227px;">Kent Scientific</td>
			<td>DCT-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sutter Instruments P30 Puller&#160;</td>
			<td>Sutter Instruments</td>
			<td>3282322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluovac Anesthesia Systems</td>
			<td>&#160;Harvard Apparatus</td>
			<td>726425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delicate Operating Scissors 4.75&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>&#160;RS-6702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Silk Black Braid 18&#34; C-1 Box 36&#160;</td>
			<td>Roboz</td>
			<td>SUT-1073-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Clip Applying Forceps 5.5&#34;</td>
			<td>Roboz</td>
			<td>&#160;RS-5410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 Clamp scissors</td>
			<td>Roboz</td>
			<td>&#160;RC-4894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding forceps&#160;</td>
			<td style="width:227px;">Fine Science Tools</td>
			<td>11031-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillary tubing&#160;</td>
			<td>&#160;FHC</td>
			<td>27-30-0</td>
			<td>Borosil 1.0mm OD x 0.75mm ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

Research

JoVE Journal

Neuroscience

8.8K Aufrufe.  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.

Serie: 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 ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

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 ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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 ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...