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 cloni...

<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 border="0" cellpadding="0" cellspacing="0" width="1106">
	<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

8.7K ビュー.  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.