Name: in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity
Uploaded: 2017-02-15T13:00:00
Description: Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

We are grateful to R. Guti&#233;rrez and A. Morales for their excellent technical assistance and to L. A. Weiss for editing. C.G.B. is funded by the Spanish Ministerio de Ciencia e Innovaci&#243;n (MICINN), FPI-BES-2012-056011. This work was funded by a grant from BBVA Foundation and SAF2014-58598-JIN (MINECO) to M. Navarrete and by a grant from the Ram&#243;n Areces Foundation and grants SAF2014-52119-R and BFU2014-55738-REDT (from MINECO) to M. Nieto.

All animal procedures were approved by the Community of Madrid Animal Care and Use Committee, in compliance with national and European legislation (PROEX 118/14; PROEX 331/15). Maintain sterile conditions during the procedure.
1. In Utero Electroporation
NOTE: This protocol for IUE is adapted from others that have been previously published5,6,7. This manuscript descr...

<ol>
	<li>Dehorter, N., 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=Tuning+of+fast-spiking+interneuron+properties+by+an+activity-dependent+transcriptional+switch.">Tuning of fast-spiking interneuron properties by an activity-dependent transcriptional switch.</a> Science. 349 (6253), 1216-1220 (2015).</li><li>Rodriguez-Tornos, F. M., 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=Cux1+Enables+Interhemispheric+Connections+of+Layer+II/III+Neurons+by+Regulating+Kv1-Dependent+Firing.">Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing.</a> Neuron. 89 (3), 494-506 (2016).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> J Vis Exp. (54), (2011).</li><li>Sonego, M., Zhou, Y., Oudin, M. J., Doherty, P., Lalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+postnatal+electroporation+and+time-lapse+imaging+of+neuroblast+migration+in+mouse+acute+brain+slices.">In vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices.</a> J Vis Exp. (81), (2013).</li><li>Baumgart, J., Baumgart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortex-,+Hippocampus-,+Thalamus-,+Hypothalamus-,Lateral+Septal+Nucleus-+and+Striatum-specific+In+Utero+Electroporation+in+the+C57BL/6+Mouse.">Cortex-, Hippocampus-, Thalamus-, Hypothalamus-,Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse.</a> J Vis Exp. (107), (2016).</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> J Vis Exp. (31), (2009).</li><li>Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. <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+electroporation+followed+by+primary+neuronal+culture+for+studying+gene+function+in+subset+of+cortical+neurons.">In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.</a> J Vis Exp. (44), (2010).</li><li>Woodworth, M. B., 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=Ctip1+Regulates+the+Balance+between+Specification+of+Distinct+Projection+Neuron+Subtypes+in+Deep+Cortical+Layers.">Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers.</a> Cell Rep. 15 (5), 999-1012 (2016).</li><li>Wickersham, I. R., 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=Monosynaptic+restriction+of+transsynaptic+tracing+from+single,+genetically+targeted+neurons.">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53 (5), 639-647 (2007).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc Natl Acad Sci U S A. 104 (3), 1027-1032 (2007).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101 (1), 16-22 (2004).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protoc. 1 (3), 1552-1558 (2006).</li><li>Bullmann, T., Arendt, T., Frey, U., Hanashima, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transportable,+inexpensive+electroporator+for+in+utero+electroporation.">A transportable, inexpensive electroporator for in utero electroporation.</a> Dev Growth Differ. , (2015).</li><li>Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+rat+visual+cortex.+I.+A+quantitative+study+of+Golgi-impregnated+pyramidal+neurons.">Maturation of rat visual cortex. I. A quantitative study of Golgi-impregnated pyramidal neurons.</a> J Neurocytol. 10 (5), 859-878 (1981).</li><li>Miller, M., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+rat+visual+cortex.+II.+A+combined+Golgi-electron+microscope+study+of+pyramidal+neurons.">Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons.</a> J Comp Neurol. 203 (4), 555-573 (1981).</li><li>Cubelos, B., 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=Cux-2+controls+the+proliferation+of+neuronal+intermediate+precursors+of+the+cortical+subventricular+zone.">Cux-2 controls the proliferation of neuronal intermediate precursors of the cortical subventricular zone.</a> Cereb Cortex. 18 (8), 1758-1770 (2008).</li><li>Kang, J. Y., Kawaguchi, D., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Control+of+a+Neuronal+Protein+Using+a+Genetically+Encoded+Unnatural+Amino+Acid+in+Neurons.">Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons.</a> J Vis Exp. (109), (2016).</li><li>Mathis, D. M., Furman, J. L., Norris, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+acute+hippocampal+slices+from+rats+and+transgenic+mice+for+the+study+of+synaptic+alterations+during+aging+and+amyloid+pathology.">Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology.</a> J Vis Exp. (49), (2011).</li><li>Maravall, M., Stern, E. A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+intrinsic+properties+and+excitability+of+layer+2/3+pyramidal+neurons+during+a+critical+period+for+sensory+maps+in+rat+barrel+cortex.">Development of intrinsic properties and excitability of layer 2/3 pyramidal neurons during a critical period for sensory maps in rat barrel cortex.</a> J Neurophysiol. 92 (1), 144-156 (2004).</li><li>Karadottir, R., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+patch-clamping+of+cells+in+brain+slices+with+immunocytochemical+labeling+to+define+cell+type+and+developmental+stage.">Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.</a> Nat Protoc. 1 (4), 1977-1986 (2006).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li></ol>

This protocol describes in detail how to label neurons of the somatosensory cortex of C75BL/6 mice in order to analyze their connectivity and their excitability. With respect to existing methods, it visualizes discriminating aspects of connectivity, such as the number of axonal branches per neuron, their precise topography, and their anatomical location. By altering the position of the electrodes, it is possible to target other neuron populations, such as the cingulate cortex (keep the same angle between the electrodes a...

The development of dendritic and axonal structures is an important facet of circuit regulation in the nervous system, including in the cerebral cortex. It plays a critical role during the selective wiring of the diverse neuronal subpopulations. A number of recent reports have shown that, in addition to connectivity, the molecular diversity of neurons is reflected by the acquisition of highly specific modes of firing. However, the mechanisms determining the excitability and connectivity of the distinct neuronal subtypes during development, as well as their degree of coordination, are still poorly understood1,2.
In vivo loss- and gain-of-function analyses allow for the study of the relationship between the expression level of specific genes and their influence in the development of the circuit. In utero electroporation (IUE) is a technique widely used to study the function of a gene of interest in specific neuronal populations and to study the overall patterns of their connectivity. However, to determine the morphological characteristics of axons and dendrites in cortical layers in living mice, it is essential to label neurons sparsely. A Cre recombination system combined with IUE can be used to mark a sparse population of neurons at a sufficiently low density to resolve the projections emitted by individual cells of the identified cortical laminas. This method labels a sufficient number of neurons per cortex to obtain quantitative data after the analysis of reasonable numbers of electroporated brains (Figure 1). This manuscript presents a method for such fine analysis of connectivity. It also presents a similar strategy to analyze, in separate experiments, the electrical properties of neurons by performing current-clamp recordings on green fluorescence protein (GFP)-electroporated cells from acute cortical slices. These protocols are versatile and can be applied to the study of the excitability and connectivity of neurons of WT and transgenic animals, and also of neurons in which losses and gains of function are introduced by additional plasmids during IUE.
Although this protocol describes the electroporation of mice at embryonic day (E)15.5, this technique can be performed at any age between E9.53 and postnatal day (P)24. While electroporation at early stages targets neurons and precursors of the thalamus and deep layers of the cortex, later-stage electroporation marks more superficial layers (e.g., E15.5 IUE targets layer II-III neurons). In summary, the combination of IUE with single-cell morphological analysis and electrophysiology is a useful tool to elucidate the molecular mechanisms underlying the enormous structural and functional diversity of neurons in the nervous system.

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			<td>pCAG-Cre</td>
			<td>Addgene</td>
			<td>13775</td>
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			<td>pCALNL-GFP</td>
			<td>Addgene</td>
			<td>13770</td>
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		<tr>
			<td>pCAG-DsRed2</td>
			<td>Addgene</td>
			<td>15777</td>
		</tr>
		<tr>
			<td>pCAG-GFP</td>
			<td>Addgene</td>
			<td>11150</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Carl Roth</td>
			<td>301.1</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
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			<td>Carprofen (Rimadyl)</td>
			<td>Pfizer GmbH</td>
			<td>1615 ESP</td>
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			<td>Isoflurane (IsoFlo)</td>
			<td>Abbott (Esteve)</td>
			<td>1385 ESP</td>
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			<td>Ketamine (Imalgene)</td>
			<td>Merial</td>
			<td>2528-ESP</td>
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			<td>Xylazine (Xilagesic)</td>
			<td>Calier</td>
			<td>0682-ESP</td>
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			<td>Povidone Iodine</td>
			<td>Meda</td>
			<td>694109.6</td>
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			<td>Eye Ointment (Lipolac)</td>
			<td>Angelini</td>
			<td>65.277</td>
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			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Gibco by Life Technologies</td>
			<td>24020-091</td>
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		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma -Aldrich</td>
			<td>P4333</td>
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			<td>Scalpel Handle #3 - 12cm</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
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			<td>Scalpel Blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
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			<td>Adson Forceps-Serrated - Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
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			<td>Hardened Fine Scissors - Straight &#160; 11 cm</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
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			<td>Scissors Mezenbaum-Nelson Curved L=14,5cm</td>
			<td>Teleflex</td>
			<td>PO143281</td>
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			<td>Thin curved tips - Style 7 Dumoxel</td>
			<td>Dumont</td>
			<td>0303-7-PO</td>
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			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
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			<td>Mathieu Needle Holder - Serrated</td>
			<td>Fine Science Tools</td>
			<td>12010-14</td>
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			<td>AutoClip Applier</td>
			<td>Braintree scientific, Inc</td>
			<td>ACS&#160;APL</td>
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			<td>9mm AutoClips</td>
			<td>MikRon Precision, Inc.</td>
			<td>205016</td>
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			<td>Sutures - Polysorb 6-0</td>
			<td>Covidien</td>
			<td>UL-101</td>
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			<td>Electric Razor&#160;</td>
			<td>Panasonic</td>
			<td>ER 240</td>
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			<td>Borosilicate glass capillaries (100mm, 1.0/0.58 Outer/Inner diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
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		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma -Aldrich</td>
			<td>A5177-5EA</td>
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		<tr>
			<td>Gauze (Aposan)</td>
			<td>Laboratorios Indas, S.A.U.</td>
			<td>C.N. 482232.8</td>
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		<tr>
			<td>Cotton Swabs (Star Cott)</td>
			<td>Albasa</td>
			<td>-</td>
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			<td>Needle 25G (BD Microlance 3)</td>
			<td>Becton, Dickinson and Company</td>
			<td>300600</td>
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			<td></td>
			<td></td>
			<td></td>
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			<td>Sucrose</td>
			<td>Sigma -Aldrich</td>
			<td>S0389</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>158127</td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100 x 20 mm</td>
			<td>Falcon</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>GFP Tag Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11122</td>
		</tr>
		<tr>
			<td>Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Electroporator ECM 830&#160;</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0002</td>
		</tr>
		<tr>
			<td>Platinum electrodes 650P 7 mm</td>
			<td>Nepagene</td>
			<td>CUY650P7</td>
		</tr>
		<tr>
			<td>Microscope&#160;for Fluorescent Imaging - MZ10F</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>VIP 3000 Isofluorane Vaporizer</td>
			<td>Matrx</td>
			<td>-</td>
		</tr>
		<tr>
			<td>TCS-SP5 Laser Scanning System</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Axiovert 200 Microscope</td>
			<td>Zeiss</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cryostat - CM 1950</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
		</tr>
		<tr>
			<td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td>
			<td>Molecular Devices</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Acquisition software (MultiClamp 700B Amplifier)</td>
			<td>Molecular Devices</td>
			<td>DD1440A</td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator + Rotating Base&#160;</td>
			<td>Sutter Instrument</td>
			<td>MP-225</td>
		</tr>
		<tr>
			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Miniature Peristaltic Pumps</td>
			<td>WPI</td>
			<td>-</td>
		</tr>
	</tbody>
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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.

To characterize the morphological changes of neurons in detail and throughout development, it is essential to label neurons sparsely. A Cre-recombinase diluted system allows for the expression of a gene of interest in a sparse population of neurons, so that only those neurons that incorporate this enzyme express GFP (Figure 1A). Using this strategy, layer II-III is targeted and labeled by IUE at E15.5. CAG-DsRed2 at 1 &#181;g/&#181;L, is co-electroporated as a control and...

Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

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.

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, ...

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Neuroscience

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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 ...

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Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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

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

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...