We thank Jacqueline Andratschke, Elena Werle, Sachi Takenaka, and Matthias Toberer for excellent technical assistance, as well as Victor Tarabykin for helpful discussions. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to S.B. (BR-2215).

All experimental procedures were approved by the Animal Welfare Committee (Regierungspr&#228;sidium T&#252;bingen) and carried out in accordance by the German Animal Welfare Act and the EU Directive 2010/63/EU.
1. In Utero Electroporation
<ol>
	<li>Microinjection needles
	<ol>
		<li>Pull borosilicate glass capillaries (outer diameter: 1.0 mm, inner diameter: 0.58 mm, length: 100 mm) into microinjection needles using a micropipette puller with a box filament (2.5 mm ...

<ol>
	<li>Evsyukova, I., Plestant, C., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+mechanisms+of+oriented+neuronal+migration+in+the+developing+brain.">Integrative mechanisms of oriented neuronal migration in the developing brain.</a> Annu Rev Cell Dev Biol. 29, 299-353 (2013).</li><li>Kwan, K. Y., Sestan, N., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+co-regulation+of+neuronal+migration+and+laminar+identity+in+the+neocortex.">Transcriptional co-regulation of neuronal migration and laminar identity in the neocortex.</a> Development. 139 (9), 1535-1546 (2012).</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><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>LoTurco, J. J., Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multipolar+stage+and+disruptions+in+neuronal+migration.">The multipolar stage and disruptions in neuronal migration.</a> Trends Neurosci. 29 (7), 407-413 (2006).</li><li>Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nat Neurosci. 7 (2), 136-144 (2004).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipolar+migration:+the+third+mode+of+radial+neuronal+migration+in+the+developing+cerebral+cortex.">Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex.</a> J Neurosci. 23 (31), 9996-10001 (2003).</li><li>Pacary, E., 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=Proneural+transcription+factors+regulate+different+steps+of+cortical+neuron+migration+through+Rnd-mediated+inhibition+of+RhoA+signaling.">Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling.</a> Neuron. 69 (6), 1069-1084 (2011).</li><li>Tabata, H., Nagata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+molecular+mechanisms+of+neuronal+migration+using+in+utero+electroporation.">Decoding the molecular mechanisms of neuronal migration using in utero electroporation.</a> Medical Molecular Morphology. 49 (2), 63-75 (2016).</li><li>Wiegreffe, C., 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=Bcl11a+(Ctip1)+Controls+Migration+of+Cortical+Projection+Neurons+through+Regulation+of+Sema3c.">Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c.</a> Neuron. 87 (2), 311-325 (2015).</li><li>John, 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=Bcl11a+is+required+for+neuronal+morphogenesis+and+sensory+circuit+formation+in+dorsal+spinal+cord+development.">Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development.</a> Development. 139 (10), 1831-1841 (2012).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal+development.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal development.</a> Sci STKE. (136), pl9 (2002).</li><li>Greig, L. C., Woodworth, M. B., Galazo, M. J., Padmanabhan, H., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+logic+of+neocortical+projection+neuron+specification,+development+and+diversity.">Molecular logic of neocortical projection neuron specification, development and diversity.</a> Nat Rev Neurosci. 14 (11), 755-769 (2013).</li><li>De Marco Garcia, N. V., 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=Subtype-selective+electroporation+of+cortical+interneurons.">Subtype-selective electroporation of cortical interneurons.</a> J Vis Exp. (90), e51518 (2014).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> J Vis Exp. (85), (2014).</li><li>Venkataramanappa, S., Simon, R., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+utero+electroporation+and+organotypic+slice+culture+of+mouse+hippocampal+tissue.">Ex utero electroporation and organotypic slice culture of mouse hippocampal tissue.</a> J Vis Exp. (97), (2015).</li><li>Simon, 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=A+dual+function+of+Bcl11b/Ctip2+in+hippocampal+neurogenesis.">A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis.</a> EMBO J. 31 (13), 2922-2936 (2012).</li><li>Youn, Y. H., Pramparo, T., Hirotsune, S., Wynshaw-Boris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dose-dependent+cortical+neuronal+migration+and+neurite+extension+defects+in+Lis1+and+Ndel1+mutant+mice.">Distinct dose-dependent cortical neuronal migration and neurite extension defects in Lis1 and Ndel1 mutant mice.</a> J Neurosci. 29 (49), 15520-15530 (2009).</li><li>Nadarajah, B., Brunstrom, J. E., Grutzendler, J., Wong, R. O., Pearlman, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+radial+migration+in+early+development+of+the+cerebral+cortex.">Two modes of radial migration in early development of the cerebral cortex.</a> Nat Neurosci. 4 (2), 143-150 (2001).</li><li>Higginbotham, H., Yokota, Y., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+analyzing+neuronal+progenitor+development+and+neuronal+migration+in+the+developing+cerebral+cortex.">Strategies for analyzing neuronal progenitor development and neuronal migration in the developing cerebral cortex.</a> Cereb Cortex. 21 (7), 1465-1474 (2011).</li><li>Stubbs, 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=Neurovascular+congruence+during+cerebral+cortical+development.">Neurovascular congruence during cerebral cortical development.</a> Cereb Cortex. 19, i32-i41 (2009).</li><li>Ayala, R., Shu, T., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trekking+across+the+brain:+the+journey+of+neuronal+migration.">Trekking across the brain: the journey of neuronal migration.</a> Cell. 128 (1), 29-43 (2007).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li></ol>

The authors have nothing to disclose.

Radial migration is a key process in neocortex development. Mutations in genes affecting different steps of this process can cause severe cortical malformations, including lissencephaly and white matter heterotopia1,2. We recently showed that Bcl11a, which is expressed in young migrating cortical projection neurons, plays a role in radial migration. We used time-lapse confocal imaging of migrating neurons in acute cortical slices of electroporated brains to direc...

The neocortex is the major site of cognitive, emotional, and sensorimotor functions. It is composed of six horizontal layers oriented in parallel to the surface of the brain. During development progenitor cells in the lateral wall of the dorsal telencephalon give rise to projection neurons that migrate radially towards the pial surface and acquire a layer type-specific neuronal identity. After being generated in the ventricular/subventricular zones (VZ/SVZ) these neurons become transiently multipolar and slow their migration. After a short stay in the intermediate zone (IZ) they switch to a bipolar morphology, attach to the radial glial scaffold, and continue radially oriented migration into the cortical plate (CP). Upon reaching their final target projection neurons detach from the radial glial processes and acquire layer-specific identity. Mutations in genes affecting different steps of neuronal migration can cause severe cortical malformation, such as lissencephaly or white matter heterotopia1,2.
In utero electroporation is a rapid and powerful technique to transfect neural progenitor cells in the developing brain of rodent embryos3,4. With this technique it is possible to knockdown and/or overexpress genes of interest in order to study their functions in developing neurons. This method has specifically helped to describe the morphological details and characterize the molecular mechanisms of the process of radial migration5,6,7,8,9. Radially migrating neurons undergo dynamic changes in cell shape, migration speed, as well as migratory direction, which require direct and continuous observation over time. Organotypic slice culture and time-lapse confocal imaging of electroporated brains allow to directly observe migrating neurons over time. Using this combined approach, it is possible to analyze distinct features of migrating neurons that cannot be investigated in fixed tissue sections of electroporated brains.
We recently applied time-lapse confocal imaging of migrating neurons in slice cultures of electroporated brains to study the role of the transcription factor B cell CLL/lymphoma 11a (Bcl11a) during cortical development10. Bcl11a is expressed in young migrating cortical neurons and we used a conditional mutant Bcl11a allele (Bcl11aflox)11 to study its functions. Electroporation of Cre recombinase together with green fluorescent protein (GFP) into cortical progenitors of Bcl11aflox/flox brains allowed us to create a mosaic mutant situation, in which only few cells are mutated in an otherwise wild-type background. In this way, it was possible to study cell-autonomous functions of Bcl11a at the single cell level. We found that Bcl11a mutant neurons display reduced speed, shifts in their speed profiles, as well as random-like orientation changes during their migration10. In the outlined protocol we describe a workflow for successful electroporation and slice culture preparation12 of mouse brains, as well as time-lapse confocal imaging of cortical slice cultures.

<table border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">isoflurane</td>
			<td style="width:171px;">Abbott Laboratories&#160;</td>
			<td style="width:111px;">506949</td>
			<td style="width:209px;">Forene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">12-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">non-absorbable surgical suture</td>
			<td style="width:171px;">Ethicon</td>
			<td style="width:111px;">K890H</td>
			<td>3/8 circle, 13 mm, taper point</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro Adson Forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11018-12</td>
			<td>serrated, length: 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">14063-09</td>
			<td>angled to side, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Mathieu Needle Holder</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">12510-14</td>
			<td>tungsten carbide, length: 14 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine tipped forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11370-40</td>
			<td>straight, 11 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Vannas T&#252;bingen Spring Scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">15005-08</td>
			<td>angled up, 9.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">ring forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11103-09</td>
			<td>OD: 3mm, ID, 2.2 mm, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HBSS (10X)</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">14180046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">L-Glutamine</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">horse serum</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">26050088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BME</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">41010026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">borosilicate glass capillaries</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:111px;">30-0016</td>
			<td>1.0 OD x 0.58 ID x 100 L mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthsesia system</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">72-6471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthetizing chamber</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluosorber filter canister</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">low melting point agarose</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:111px;">16520100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">vibrating blade microtome</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">VT1200 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluorescence stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M205 FA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">inverted fluorescence tissue culture microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">DM IL LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">confocal laser scanning microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">TCS SP5II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">hybrid detector</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">HyD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">objective, 40x/0.60 NA</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">11506201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">microscope temperature control system</td>
			<td style="width:171px;">Life Imaging Services</td>
			<td style="width:111px;">Cube, Brick &#38; Box</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">cell culture insert</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:111px;">PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microgrinder</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:111px;">EG-45</td>
			<td>use 38&#176; angle for beveling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microinjector</td>
			<td style="width:171px;">Parker Hannifin&#160;</td>
			<td>052-0500-900</td>
			<td style="width:209px;">Picospritzer III</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">carprofen</td>
			<td style="width:171px;">Pfizer Animal Health</td>
			<td style="width:111px;">NDC 61106-8507</td>
			<td>Rimadyl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">emdedding mold</td>
			<td style="width:171px;">Polysciences</td>
			<td style="width:111px;">18986-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">endotoxin-free plasmid maxi kit</td>
			<td style="width:171px;">Qiagen</td>
			<td style="width:111px;">12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fast green</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">F7252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">laminin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">poly-L-lysine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HEPES</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">D-glucose</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">magensium sulfate</td>
			<td>Sigma</td>
			<td style="width:111px;">M2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">sodium bicarbonate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">square wave electroporator</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY21EDIT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">tweezers with 5 mm platinum disk electrodes</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micropipette puller</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">box filament</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">FB255B</td>
			<td>2.5 mm x 2.5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micro-spoon spatula</td>
			<td style="width:171px;">VWR</td>
			<td style="width:111px;">231-0191</td>
			<td>185 mm x 5 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">glass bottom dish, 50 mm</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:111px;">FD5040-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Previously, we have shown that genetic deletion of Bcl11a by in utero electroporation impairs radial migration of late-born upper-layer projection neurons10. Electroporation of a DNA plasmid vector containing Cre-IRES-GFP efficiently deleted Bcl11a in conditional Bcl11aflox/flox brains11. When we analyzed E14.5 electroporated brains three days after the electroporation, most control neu...

Watch this Scientific Journal Video about Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation at JoVE.com

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

The overall goal of this procedure is to directly observe radially migrating neurons in organotypic slice culture prepared from electroporated embryonic brain by time-lapse confocal microscopy. This method can help study key aspects of neocortex development. It allows investigation of the molecular mechanisms involved in neuron polarization and the radial migration of neurons to their final position within the brain.The main advantage of this technique is the dynamic properties of migrating neurons can be analyzed including speed profiles, average migration speed as well changes of migratory direction. Begin by placing a properly anesthetized pregnant mouse in the supine position on a warming plate. Check for the absence of the pedal reflex.And then, carefully cover the eyes with petroleum jelly to prevent them from drying. Next, gently spread out the limbs and fix them to the warming plate with surgical tape. Sterilize the abdomen by swabbing with 70%ethanol followed by iodine solution, then place sterile gauze, in which a cut has been made for the abdominal incision, over the abdomen.Moisten the gauze with bacteriostatic sodium chloride solution. After reassessing the development of surgical anesthesia, by loss of the pedal reflex, use serrated micro Adson forceps and angled fine scissors to cut the skin approximately 1.5 centimeters along the midline of the abdomen. Then, cut the underlying muscle along the linea alba.Grasp the uterine horn between embryos with ring forceps and gently place it onto the moistened gauze without perturbing the placentas or supply vessels. Then, carefully position an embryo to give a clear view of the lateral ventricle which presents as a crescent-shaped shadow parallel to the sagittal sinus. The injection site lies in the middle of a line between the pigmented eye and the confluence of sinuses where the sagittal sinus branches to two transfer sinuses.Once identified, push the microinjection needle through the uterine wall and into the lateral ventricle. Next, use a microinjector operated with a foot pedal to inject one to two microliters of DNA solution with five to 10 pulses. Successful injection can be monitored by the colored DNA solution which should fill the ventricular system.Then, place tweezers-type electrodes so that the positive terminal is on the same side as the injected ventricle and the negative terminal is on the opposite side of the injected ventricle below the ear of the embryo's head. Moisten the electroporation site with a few drops of bacteriostatic sodium chloride solution and apply five electrical current pulses of 50-millisecond duration with 950-millisecond intervals. Bubbles at the negative electrode indicate electrical current.60 to 90 milliamperes are usually sufficient for successful electroporation. Lower currents will not efficiently transfect neurons whereas higher currents may lead to embryonic death. After repeating the procedure for every embryo of the first uterine horn, gently place it back into the abdominal cavity.After suturing the muscle layer and skin, carefully disinfect with iodine solution and gently remove the petroleum jelly from the eyes using cellulose swabs. Place the mouse under an infrared lamp until it wakes up. After euthanizing the pregnant female using an approved method, remove the uterus containing the embryos and place it into a 10-centimeter Petri dish with ice-cold complete HBSS.From this point onward, keep all solutions, embryos and brains on ice. While working under a stereomicroscope, use fine-tipped forceps and a pair of Vannas Tubingen spring scissors to separate each embryo from the uterus. Transfer the embryos to another Petri dish containing ice-cold complete HBSS.Make an incision at the level of the brainstem and cut along the sagittal midline. Peel away the skin and cartilage covering the brain. Then, cut off the brainstem right behind the hemispheres and remove the brain from the skull.Transfer the brain with a micro spoon spatula to a 12-well plate containing ice-cold complete HBSS. Collect all brains from the litter in the 12-well plate. Then, use a fluorescent stereomicroscope to screen the brains in the 12-well plate for brightness of florescence as well as the size of the electroporated region.Select two to four brains with bright fluorescent signals and the presumptive somatosensory cortex for further processing. Then, pour molten 3%low-melting-point agarose solution into a peel-away disposable embedding mold. Use a micro spoon spatula to remove the brain from the 12-well plate and carefully drain excess complete HBSS from around the brain using fine tissue paper.Place the brain gently into the agarose solution. Then, use a 20-gauge needle to push it to the bottom of the mold and carefully adjust its position. For coronal sections, orient the brain with the olfactory bulbs pointing upwards.Keep the mold on ice until the agarose solution is solidified then proceed to sectioning the brain. Use a clean razor blade to trim away excess agarose around the brain leaving approximately one millimeter of agarose on all sides except for the olfactory bulbs where two to three millimeters of agarose should be left. After applying a small amount of cyanoacrylate glue to a specimen, fix the trimmed agarose block with the olfactory bulbs pointing downwards.Transfer the specimen stage to the slicing chamber of a vibrating blade microtome containing ice-cold complete HBSS and position the brain with its dorsal side towards the blade. Cut 250 micron-thick brain slices containing the electroporated region with a low to moderate amplitude and a very slow sectioning speed. Usually four to six slices with bright fluorescent signal are obtained from each successfully electroporated brain.Grasp the agarose rim of one section with fine-tipped forceps and pull the slice onto a bent micro spoon spatula. In this manner, carefully transfer the brain slices to a six-well plate containing ice-cold complete HBSS. Moisten laminin-coated membrane inserts with 100 microliters of complete HBSS.Then, carefully transfer the slices onto the membranes using the bent micro spoon spatula and forceps. Gently push the slice from the spatula onto the membrane, then position using forceps. Continue to transfer the remaining slices.Up to five slices can be placed on one membrane insert. Remove excess complete HBSS with a pipette. Then, with the help of forceps, carefully place the membrane inserts with the slices into a six-well plate containing 1.8 milliliters of sliced culture medium.Select a brain slice for imaging by viewing the slices through an inverted microscope. Choose a slice with bright single neurons in the upper SVZ migrating radially towards the pile surface of the slice. The presence of fine fluorescent processes of radial glial cells that span the entire conjugate plate indicates an intact radial glial scaffold which is used by migrating conjugate neurons.Transfer the membrane insert with a selected slice into a 50-millimeter-diameter glass-bottomed dish containing two milliliters of slice culture medium. Place the dish into the climate chamber of the confocal microscope. Set the resolution to 512 by 512 pixels.Increase the scan speed from 400 hertz to 700 hertz to increase the frame rate from about 1.4 to about 2.5 frames per second. Use no more than two times averaging. Define a Z-stack through the electroporated region with the step size of 1.5 microns.Start the time-lapse series by taking a Z-stack every 30 minutes. The settings described allow for sufficient resolution and brightness of the image while keeping photo damage low during acquisition. This movie shows E16.5 cortical neurons migrating from the ventricular subventrical zones to the cortical plate.In the Bcl11a floxed conditional transgenic after electroporation of the IRES-GFP control plasmid on embryonic day 14.5. The movie is comprised of 108 frames at the rate of five frames per second. The scale bar represents 100 microns.Electroporation of a DNA plasmid vector containing Cre-IRES-GFP affected cortical neuron migration in conditional Bcl11a floxed brains. As seen here, very few neurons move past the intermediate zone to reach the cortical plate. This movie shows the polarization of cortical neurons from a GFP-labeled control on the left compared to neurons from a Bcl11a mutant on the right.These animated traces of representative control in Bcl11a mutant neurons were obtained from time-lapse series of E16.5 slice cultures with one-hour-interval resolution. Again, data from the control brain is shown on the left and data from the Cre-IRES-GFP electroporated brain is shown on the right. As seen from this collection of representative traces from control and Bcl11a mutant slice cultures, the Bcl11a mutant neurons frequently undergo repetitive phases of reduced migration speed and randomly changed orientation as marked by the red arrowheads.Speed profiles can be calculated from traces of migrating neurons. As seen here, a greater proportion of Bcl11a mutant neurons have slower migration speeds compared to controls. The deviation angle may be calculated from the trace data.As seen from this histogram, the Bcl11a mutant neurons represented by the black bars exhibit greater angles of deviation compared to controls. Once mastered, this technique can be done in less than two hours each for the in utero electroporation and the preparation of organotypic brain slice culture. This procedure can easily be adapted to perform fractional analysis of genes of interest in radially migrating cortical neurons by gain and loss of function as well as vascular experiments.After watching this video, you should have a good understanding of how to directly observe radially migrating neurons in organotypic slice culture prepared from electroporated embryonic brains by time-lapse confocal microscopy.

time-lapse-confocal-imaging-migrating-neurons-organotypic-slice

Research

JoVE Journal

Neuroscience

10.0K visualizações.  Ulm University. The overall goal of this procedure is to directly observe radially migrating neurons in organotypic slice culture prepared from electroporated embryonic brain by time-lapse confocal microscopy. This method can help study key aspects of neocortex development. It allows investigation of the molecular mechanisms involved in neuron polarization and the radial migration of neurons to their final position within the brain.The main advantage of this technique is the dynamic properties of migr...