Nous sommes reconnaissants à Lihong Yin pour l&#39;assistance technique. NVD est récipiendaire d&#39;un Prix du jeune chercheur NARSAD et est également soutenu par des subventions du NIH (5 K99 MH095825-02). La recherche dans le laboratoire Fishell est soutenu par l&#39;Institut national de la santé, Institut national de santé mentale (5 R01 MH095147-02, 5 R01 MH071679-09), l&#39;Institut national des troubles neurologiques et des maladies (5 R01 NS081297-02, 1 P01 NS074972 -01A1) et la Fondation Simons.

Tous les animaux ont été traités en conformité avec les règlements et les directives du soin et l&#39;utilisation des animaux Commission institutionnelle de la NYU School of Medicine. Les souches de souris Webster souris femelles suisses fournies par TACONIC ont été utilisés pour ces expériences. Afin de cibler spécifiquement les interneurones de la couche superficielle, embryons E15.5 ont été utilisés. Remarque: Le plasmide utilisé dans ce travail (Dlx5 / 6.eGFP plasmide 3 ug / ul) ont été générés en utilisant des techniques de clonage standard. L&#39;ADNc de eGFP a été clone dans un Dlx5 / Pmin-6-polyA plasmide. Ce plasmide est disponible sur demande (demarn02@nyumc.org). 1 Préparation de microinjection Pipettes <ol><li> Définir l&#39;extracteur de chaleur # 2-860. </li><li> Placer et fixer le capillaire de verre près du filament. Vérifiez que le diamètre extérieur (OD) de la pipette est d&#39;environ 30 um. </li><li> Appuyez sur le bouton &quot;tirer&quot;. </li><li> Retirez délicatement le capillaire. La partie inférieure est l&#39;aiguille. Jeter la partie supérieure. </li></ol> 2. Procédure anesthésie <ol><li> Préparer une chambre d&#39;isoflurane contenant. Remarque: L&#39;isoflurane est l&#39;anesthésique privilégié en raison de son action rapide et une faible incidence d&#39;effets secondaires. Pentobarbital est une autre option; cependant, la tolérance de ce médicament est variable et peut conduire à un taux de mortalité supérieur à l&#39;isoflurane. </li><li> Régler le débit de l&#39;isoflurane à 4-5% dans 0,5-1 L / min O 2. </li><li> Assurez-vous que la valve de la chambre est ouverte alors que la vanne de l&#39;adaptateur de tête est fermé. </li><li> Placez la souris enceinte dans la chambre. </li><li> Laissez la souris pour aller sous l&#39;effet de l&#39;anesthésie. Cela prendra environ 2-3 min. Vérifiez le taux de respiration. Si la souris commence hyperventilation, réduire la dose d&#39;isoflurane. </li><li> Après la souris est anesthésiée, retirez rapidement à partir de la chambre. Placez-le face ventrale sur le coussin chauffant et insert la tête dans le masque qui va continuer à fournir de l&#39;isoflurane dans toute la chirurgie. Assurez-vous de passer le flux d&#39;isoflurane de la chambre à la tubulure reliant le morceau d&#39;adaptateur de tête. Mettre une goutte de lubrifiant oculaire dans chaque œil et fermez les yeux. </li><li> Régler le coussin chauffant à 36 ° degrés C pour réduire au minimum l&#39;hypothermie. </li><li> Vérifier l&#39;absence de réflexes du pied et de la queue en pinçant les pattes arrière et la queue. </li></ol> 3. chirurgie <ol><li> Nettoyer la peau qui recouvre l&#39;abdomen avec 70% d&#39;éthanol. </li><li> Utilisez une pince pour tirer la peau vers le haut. Après chaque intervention, laver tous les instruments avec du détergent et de l&#39;eau, et stérilisé à 72 ° CO / N. </li><li> Faire une petite incision verticale (environ 2 cm) le long de la ligne médiane de départ à mi-chemin entre les troisième et quatrième paires de glandes mammaires. Utilisez des ciseaux fins de chirurgie. Prenez soin de ne pas endommager la paroi abdominale. </li><li> Séparer la peau de la douceur péritoine. </li><li> Visualiser les artèreset les veines sur la paroi abdominale. Faire une autre incision verticale de 2 cm à travers le péritoine (en évitant les vaisseaux) pour exposer la cavité abdominale. </li><li> Serrer la peau et la paroi abdominale de chaque côté en évitant le serrage des artères. Remarque: Si les artères sont accidentellement percés, sacrifier immédiatement l&#39;animal en effectuant dislocation cervicale ou surdosage d&#39;isoflurane. </li><li> Pré chauffer la PBS stérile à 37 ° C. Couvrir les colliers avec du PBS humides Kim lingettes. </li><li> Hydrater la cavité abdominale exposée avec du PBS. Répétez cette étape plusieurs fois pendant la chirurgie. </li><li> En utilisant des pinces rondes, tirez doucement sur la chaîne embryonnaire (E15.5) de la cavité. Laissez la chaîne reste sur les lingettes humides. Commencer par exposition de la moitié de la première utérus. Une fois que les embryons qui s&#39;y trouvent sont électroporation, le ramener à l&#39;intérieur, puis de travailler sur le second semestre. </li></ol> 4. électroporation <ol><li> Continuer à utiliser des forceps rondes de manipuler les embryons. </li><li> Visualize les ventricules latéraux. Les ventricules latéraux courent le long de la ligne médiane (figure 1b). </li><li> Ajouter vert Fast (stock: 10% p / v) de la solution d&#39;ADN pour un mélange 01:20 de visualiser pendant l&#39;injection. Préparer la solution d&#39;ADN par dissolution du culot obtenu à partir d&#39;un protocole de purification maxi-prep standard dans de l&#39;eau distillée. </li><li> Utilisation micropipettes pré tirés avec un piston pour injecter 1 ml de solution d&#39;ADN (Dlx5 / 6-eGFP, 3 ug / ul) avec Fast Green. Couper l&#39;extrémité de la micropipette avec une pince amende # 5 (Dumont) de laisser 6 mm de longueur à partir du début de l&#39;épaule à l&#39;extrémité de la pointe de l&#39;aiguille avant de charger la pipette. Maintenir la tête de l&#39;embryon avec des pincettes rondes. Entrez le cerveau avec la pipette à un angle de 45 ° en visant le ventricule latéral situé à proximité de la ligne médiane. Si l&#39;aiguille pénètre dans le ventricule, se rétracter lentement la pipette que vous utilisez le piston pour injecter l&#39;ADN. Le ventricule doit devenir vert lorsque lesolution commence à le remplir. À ce stade, arrêter de bouger la pipette et injecter 1 ml d&#39;ADN. Doucement, retirer la pipette. </li><li> Réglez le électroporateur de CUY21 cinq 45V impulsions de 50 ms espacées de 950 ms. </li><li> Utilisez 5 mm électrodes des palettes pour les embryons de E15.5. Plonger les électrodes dans du PBS pour assurer une transmission efficace du courant. </li><li> Placer les palettes d&#39;électrodes autour de la tête de l&#39;embryon de la palette sur le ventricule positif contenant la solution d&#39;ADN. Baisser légèrement la palette positif en ce qui concerne la négative à créer un courant ventral par les éminences ganglionnaires. Cette manipulation permet de minimiser l&#39;expression ectopique dans les cellules pyramidales. </li><li> Après avoir placé les électrodes, délivrer une impulsion en appuyant sur la pédale de fois. </li><li> Retirez les électrodes et essuyez-les. Répéter les étapes 4.6 à 4.9 de chaque embryon. </li><li> Après électroporation tous les embryons, verser 3 ml de PBS dans la cavité abdominale et les retourner à la cavité abdominale. </li><li>Première suture la paroi abdominale avec une aiguille courbe et 5-0 suture de soie et la peau. Appliquer la lidocaïne (4%) sur la plaie. Administrer 120 mg / kg de l&#39;aspirine péritoine intra pour l&#39;analgésie. Remarque: L&#39;ensemble de la procédure doit être effectuée en 20 minutes ou moins. Il est recommandé que les débutants ne électroporer quelques embryons de garder le temps de fonctionnement court. Chirurgies prolongé diminue le taux de survie des embryons. </li><li> Retirer l&#39;animal de la tubulure d&#39;anesthésie et permet la récupération sur le coussin chauffant sur un papier chiffon. </li><li> Animaux Retour à la cage, le garder sur le coussin chauffant à 37 ° C et de surveiller l&#39;état de santé. Les animaux tolèrent généralement la chirurgie bien et commencent à se déplacer lentement peu de temps après leur retrait de la tubulure de l&#39;anesthésie. Si le saignement excessif ou de la léthargie est observé, sacrifier l&#39;animal immédiatement en effectuant IACUC approuvé les procédures d&#39;euthanasie tels que la dislocation cervicale ou surdosage d&#39;anesthésie. </li><li> Cage Retour à la soutenancerium. Les femelles donnent naissance naturellement. </li></ol>

<ol>
	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. W., Wray, S., Mahon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472, 351-355 (2011).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> Journal of visualized experiments : JoVE. (31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

Les auteurs n&#39;ont rien à divulguer.

Limites de la technique Bien que cette technique permet l&#39;analyse de cellule autonome de processus cellulaires, il n&#39;est pas adapté à l&#39;analyse de la population. Les électroporations sont très rares avec moins d&#39;un millier de cellules par électroporation par le cerveau. En conséquence, la technique ne peut pas être utilisé pour évaluer les conséquences sur le comportement résultant de la manipulation génétique des interneurones CGE dérivés. <p class="jove_c...

La majeure partie des interneurones GABAergiques corticaux proviennent de deux structures embryonnaires transitoires nommés les éminences ganglionnaires médianes et caudales (MGE et CGE respectivement) 4. Parvalbumine et la somatostatine exprimer interneurones proviennent de la MGE alors Calretinin (Cr), Vasointestinal peptide (VIP) et Reelin (Re) exprimant interneurones proviennent de la CGE. Ces sous-types interneurones peuvent être distingués par leur date de naissance. MGE sous-types dérivés sont nés entre le jour embryonnaire 9.5 (de E9.5) et E16,5 5,6. En revanche, les interneurones CGE dérivés sont nés de E12.5 par a 18,5 avec leur production culminant à E15.5 6. Le ciblage génétique de cette population née fin, cependant, reste insaisissable. Les distale moins (Dlx) gènes murins sont exclusivement exprimés dans le cerveau antérieur ventral développement 3. Interneurones GABAergiques et les neurones de projection du striatum mais cellules pyramidales corticales pas exprimer Dlx1, 2,5, et 6 gènes à des stades de développement précoces 3. En effet, les gènes Dlx sont exprimés dans la zone sous-ventriculaire MGE et CGE (SVZ) dans tous les progéniteurs GABAergiques. L&#39;expression de ces gènes se limite à sélectionner les sous-types de stades post-mitotiques 9.7. Preuves expérimentales antérieures ont montré que l&#39;élément Dlx5 / 6 activateur permet le ciblage sélectif de lignées GABAergiques dans des modèles de souris transgéniques 2. Nous avons testé l&#39;utilisation de l&#39;un de ces éléments amplificateurs dans le contexte de l&#39;expression épisomique dans le cerveau de souris en développement. Nous avons cloné sous l&#39;élément Dlx5 / 6 amplificatrice avec un promoteur minimal et la protéine fluorescente verte améliorée (EGFP) dans un bluescript (BS) squelette plasmidique (Figure 1). Nous avons introduit le plasmide au moyen d&#39;électroporation in utero à E15.5 pour cibler sélectivement Cr, VIP et sous-types République 3,8,10. Notre technique permet clairsemée électroporation, ce qui facilitela reconstruction des caractéristiques morphologiques des cellules de singe. En outre, les niveaux exceptionnellement élevés d&#39;expression de gènes dans les neurones corticaux GABAergiques permet pour des études fonctionnelles. Nous avons effectué la perte et le gain d&#39;études de fonction à l&#39;aide de plusieurs gènes de type sauvage et négatifs dominants 11.

<table border="0" cellpadding="0" cellspacing="0" width="1106">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Electroporator&#160; with pedal</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY21</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mm paddle electrodes&#160;</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad&#160;</td>
			<td style="width:227px;">Kent Scientific</td>
			<td>DCT-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sutter Instruments P30 Puller&#160;</td>
			<td>Sutter Instruments</td>
			<td>3282322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluovac Anesthesia Systems</td>
			<td>&#160;Harvard Apparatus</td>
			<td>726425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delicate Operating Scissors 4.75&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>&#160;RS-6702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Silk Black Braid 18&#34; C-1 Box 36&#160;</td>
			<td>Roboz</td>
			<td>SUT-1073-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Clip Applying Forceps 5.5&#34;</td>
			<td>Roboz</td>
			<td>&#160;RS-5410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 Clamp scissors</td>
			<td>Roboz</td>
			<td>&#160;RC-4894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding forceps&#160;</td>
			<td style="width:227px;">Fine Science Tools</td>
			<td>11031-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillary tubing&#160;</td>
			<td>&#160;FHC</td>
			<td>27-30-0</td>
			<td>Borosil 1.0mm OD x 0.75mm ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile PBS</td>
			<td>Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtype-selective electroporation of cortical interneurons

L&#39;étude de système nerveux central (SNC) maturation repose sur le ciblage génétique des populations neuronales. Cependant, la tâche de restreindre l&#39;expression de gènes d&#39;intérêt à sous-types neuronaux spécifiques s&#39;est avérée remarquablement difficile en raison de la rareté relative des éléments spécifiques de promoteurs. Interneurones GABAergiques constituent une population neuronale avec une grande diversité morphologique et génétique. En effet, plus de 11 sous-types différents d&#39;interneurones GABAergiques ont été caractérisées dans le cortex de souris 1. Nous présentons ici un protocole adapté pour le ciblage sélectif des populations GABAergiques. Nous avons obtenu un ciblage sélectif de sous-type des interneurones GABAergiques en utilisant l&#39;élément activateur de la transcription à homéoboîte facteurs Dlx5 et Dlx6, des homologues de la moins distale (Dll) gène Drosophila 2,3, pour entraîner l&#39;expression de gènes spécifiques par le biais électroporation in utero.

Nous avons adapté la technique d&#39;électroporation in utero pour atteindre type de cellule ciblage spécifique des neurones échéance. Pour conduire l&#39;expression de eGFP dans les interneurones CGE dérivés, nous avons utilisé l&#39;élément amplificateur de Dlx5 / 6 et limité nos injections à E15.5, la scène où la majorité des interneurones CGE dérivés sont générés. Nous avons effectué l&#39;analyse à P8 et P15 11 (figures 1 et 2). No...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sous-sélective électroporation corticaux Interneurones

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.8K vues.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Vidéo: Subtype-selective Electroporation of Cortical Interneurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero électroporation suivie par la culture neuronale primaire pour étudier la fonction des gènes dans un sous-ensemble des neurones corticaux

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Recherche

Neurosciences

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En électroporation in vivo de la rétine de souris en développement

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolement et culture des astrocytes corticaux de souris

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

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

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

Néonatale électroporation zone sous-ventriculaire

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

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero électroporation et explants hémisphère: Une méthode simple expérimental pour les études du développement cortical précoce

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Néonatale Pial électroporation de surface

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

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

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

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

De cellules entières de patch-clamp enregistrements de Morphologically- et interneurones hippocampiques neurochemically identifiés

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

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Un guide pour<em&gt; In vivo</em&gt; Simple unité d&#39;enregistrement de Optogenetically identifié corticale inhibitrice Interneurones

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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

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

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

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

Méthode électrophysiologique de réponses d&#39;enregistrement intracellulaire tension de<em&gt; Drosophila</em&gt; Photorécepteurs et Interneurones à Light Stimuli<em&gt; In Vivo</em

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

Visualization of Cortical Modules in Flattened Mammalian Cortices

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

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

Visualisation des Modules corticales dans cortex mammifère aplatie

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

Sous-sélective électroporation corticaux Interneurones

Résumé

Explorer plus de vidéos

Chapitres dans cette vidéo