Name: time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation
Uploaded: 2017-07-25T12:30:00
Description: 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

Nous remercions Jacqueline Andratschke, Elena Werle, Sachi Takenaka et Matthias Toberer pour une excellente assistance technique, ainsi que Victor Tarabykin pour des discussions utiles. Ce travail a été soutenu par une subvention de la Deutsche Forschungsgemeinschaft à SB (BR-2215).

Toutes les procédures expérimentales ont été approuvées par le Comité du bien-être animal (Regierungspräsidium Tübingen) et effectuées conformément à la loi allemande sur le bien-être animal et à la directive européenne 2010/63 / UE. 1. Dans Utero Electroporation <ol><li> Aiguilles de micro-injection <ol><li> Tirez les capillaires en verre de borosilicate (diamètre extérieur: 1,0 mm, diamètre intérieur: 0,58 mm, longueur: 100 mm) dans des aiguil...

<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>

La migration radiale est un processus clé dans le développement de neocortex. Les mutations dans les gènes affectant différentes étapes de ce processus peuvent provoquer des malformations corticales sévères, y compris lissencéphalie et hétérotopie blanche 1 , 2 . Nous avons récemment montré que Bcl11a, qui s&#39;exprime dans les jeunes neurones de projection corticale migratoire, joue un rôle dans la migration radiale. Nous avons utilisé une imager...

Le néocortex est le principal site de fonctions cognitives, émotionnelles et sensoriomotrices. Il est composé de six couches horizontales orientées parallèlement à la surface du cerveau. Au cours du développement, les cellules progénitrices dans la paroi latérale du télencephalon dorsal donnent lieu à des neurones de projection qui migrent radialement vers la surface du pial et qui acquièrent une identité neuronale spécifique au type de couche. Après avoir été généré dans les zones ventriculaires / subventriculaires (VZ / SVZ), ces neurones deviennent transitoirement multipolaires et ralentissent leur migration. Après un court séjour dans la zone intermédiaire (IZ), ils passent à une morphologie bipolaire, attachent à l&#39;échafaudage radial et poursuivent une migration orientée radialement vers la plaque corticale (CP). Après avoir atteint leur cible finale, les neurones se détachent des processus de glande radiale et acquièrent une identité spécifique de la couche. Les mutations dans les gènes affectant différentes étapes de la migration neuronale peuvent causer de graves malformations corticales, telles que lissencL&#39;hétérotopie de l&#39;ephalie ou de la matière blanche 1 , 2 . L&#39; électroporation in utero est une technique rapide et puissante pour transfecter des cellules progénitrices neurales dans le cerveau en développement d&#39;embryons de rongeurs 3 , 4 . Avec cette technique, il est possible de supprimer et / ou de surexprimer les gènes d&#39;intérêt afin d&#39;étudier leurs fonctions dans le développement des neurones. Cette méthode a spécifiquement aidé à décrire les détails morphologiques et à caractériser les mécanismes moléculaires du processus de migration radiale 5 , 6 , 7 , 8 , 9 . Les neurones qui migrent radialement subissent des changements dynamiques dans la forme de la cellule, la vitesse de migration, ainsi que la direction migratoire, qui nécessitent une observation directe et continue au fil du temps. Cultivé en tranches organotypiquesL&#39;imagerie confocale à temps réel et temporelle des cerveaux électroporés permet d&#39;observer directement les neurones migrateurs dans le temps. En utilisant cette approche combinée, il est possible d&#39;analyser des caractéristiques distinctes des neurones migrateurs qui ne peuvent pas être étudiées dans des coupes fixes de tissus de cerveaux électroporés. Nous avons récemment appliqué l&#39;imagerie confocale temporelle des neurones migrateurs dans les cultures en tranche de cerveaux électroporés pour étudier le rôle du facteur de transcription CLL / lymphome 11a (Bcl11a) pendant le développement cortical 10 . Bcl11a s&#39;exprime chez les jeunes neurones corticaux migrateurs et nous avons utilisé un allèle Bcl11a mutant conditionnel ( Bcl11a flox ) 11 pour étudier ses fonctions. L&#39;électroporation de Cre recombinase avec la protéine fluorescente verte (GFP) dans les progéniteurs corticaux des cerveaux Bcl11a flox / flox nous a permis de créer une situation de mutant de mosaïque, dans laquelle seulement quelques cellules sont mutées dans unSinon un arrière-plan de type sauvage. De cette façon, il était possible d&#39;étudier les fonctions autonomes de cellules de Bcl11a au niveau de cellule unique. Nous avons constaté que les neurones mutants Bcl11a affichent une vitesse réduite, des changements dans leurs profils de vitesse, ainsi que des changements d&#39;orientation aléatoires pendant leur migration 10 . Dans le protocole décrit, nous décrivons un flux de travail pour l&#39;élimination réussie de l&#39;électroporation et la préparation des cultures en tranche 12 du cerveau de la souris, ainsi que l&#39;imagerie confocale temporelle des cultures corticales en tranches.

<table border="0" cellpadding="0" cellspacing="0" width="771">
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			<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

L&#39; électroporation in utero est une approche rapide et puissante pour étudier le processus de migration radiale dans le cortex cérébral du développement d&#39;embryons de souris. Il a aidé à décrire les différentes étapes de la migration radiale et à caractériser les mécanismes moléculaires qui contrôlent ce processus. Pour analyser directement et dynamiquement les neurones migrateurs, ils doivent être tracés dans le temps. Ce protocole décrit un flux de travail qui combine l&#39; électroporation utérotique avec la culture de tranche organotypique et l&#39;imagerie confocale temporelle, ce qui permet un examen direct et une analyse dynamique des neurones corticaux migrant radialement. En outre, une caractérisation détaillée des neurones migrateurs, tels que la vitesse de migration, les profils de vitesse, ainsi que les changements d&#39;orientation radiale, est possible. La méthode peut facilement être adaptée pour effectuer des analyses fonctionnelles de gènes d&#39;intérêt pour la migration radiale des neurones corticaux par perte et gain de fonction ainsi que des expériences de sauvetage. Laps de tempsL&#39;imagerie des neurones migrateurs est une technique de pointe qui, une fois établie, est un outil puissant pour étudier le développement du cortex cérébral dans les modèles de souris des troubles de la migration neuronale.

Auparavant, nous avons montré que la suppression génétique de Bcl11a par l&#39; électroporation in uuer altère la migration radiale des neurones de projection de la couche supérieure tardive 10 . L&#39;électroporation d&#39;un vecteur plasmidique d&#39;ADN contenant Cre-IRES-GFP supprimé efficacement Bcl11a dans des cerveaux BCL11a flox / flox conditionnels 11...

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

Ce protocole fournit des instructions pour l&#39;observation directe des neurones corticaux migrant radialement. L&#39; électroporation in utero , la culture de tranche organotypique et l&#39;imagerie confocale temporelle sont combinés pour étudier directement et dynamiquement les effets de la surexpression ou la régulation négative des gènes d&#39;intérêt dans les neurones migrateurs et d&#39;analyser leur différenciation au cours du développement.

L&#39;imagerie confocale temporelle des neurones migrateurs dans la culture de tranches organotypiques du cerveau embryonnaire de souris en utilisant<em&gt; Dans Utero</em&gt; 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.

Research

JoVE Journal

Neuroscience

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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

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

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