Wir sind dankbar, dass Lihong Yin für technische Unterstützung. NVD ist ein Empfänger einer NARSAD Young Investigator Award und wird auch durch Zuschüsse aus NIH (5 K99 MH095825-02) unterstützt. Forschung im Labor Fishell wird durch das National Institute of Health, National Institute of Mental Health (5 R01 MH095147-02, 5 R01 MH071679-09), National Institute of Neurological Disorders and Stroke (5 R01 NS081297-02 unterstützt, 1 P01 NS074972 -01A1) und der Simons Foundation.

Alle Tiere wurden in Übereinstimmung mit den Vorschriften und Richtlinien des Institutional Animal Care und Use Committee der New York University School of Medicine behandelt. Mausstämme Swiss Webster-Mäuse von weiblichen TACONIC bereitgestellt wurden für diese Experimente verwendet. Um gezielt oberflächliche Schicht Inter wurden E15.5 Embryonen verwendet. Hinweis: Die in dieser Arbeit (Dlx5 / 6.eGFP Plasmid 3 ug / ul) wurde unter Verwendung von Standardtechniken erzeugt Klonen verwendete Plasmid. Die eGFP-cDNA wurde in einer Dlx5 / 6-Pmin-Poly-A-Plasmid kloniert. Dieses Plasmid ist auf Anfrage erhältlich (demarn02@nyumc.org). 1. Herstellung der Mikroinjektion Pipetten <ol><li> Stellen Sie den Abzieher Heat # 2-860. </li><li> Ort und sichern die Glaskapillare in der Nähe des Filaments. Überprüfen Sie, dass der Außendurchmesser (OD) der Pipette ist in etwa 30 um. </li><li> Drücken Sie die Taste &quot;Pull&quot;. </li><li> Die Kapillare vorsichtig entfernen. Der untere Teil ist die Nadel. Entsorgen Sie den oberen Teil. </li></ol> 2. Anästhesieverfahren <ol><li> Bereiten Sie eine Isofluran enthaltenden Kammer. Hinweis: Isofluran ist die bevorzugte Betäubung, durch die schnelle Wirkung und geringe Inzidenz von Nebenwirkungen. Pentobarbital ist eine weitere Option; jedoch ist die Toleranz dieser Droge variabel und kann auf eine höhere Mortalität als Isofluran führen. </li><li> Stellen Sie den Durchfluss von Isofluran zu 4-5% in 0,5-1 l / min O 2. </li><li> Stellen Sie sicher, dass das Ventil für die Kammer ist offen, während die Ventiladapter für den Kopf ist geschlossen. </li><li> Platzieren Sie die Maus schwanger in der Kammer. </li><li> Lassen Sie die Maus, um unter der Wirkung der Narkose zu gehen. Dies wird etwa 2-3 Minuten dauern. Überprüfen Sie die Atemfrequenz. Wenn die Maus beginnt zu hyperventilieren, senken Sie die Dosis von Isofluran. </li><li> Nachdem die Maus betäubt, umgehend entfernen es aus der Kammer. Legen Sie sie Bauchseite auf dem Heizkissen und ichnsert den Kopf in der Maske, die weiterhin in der gesamten Chirurgie Isofluran versorgen wird. Stellen Sie sicher, um den Fluss von Isofluran aus der Kammer in den Schlauch verbindet den Kopf Adapterstück zu wechseln. Geben Sie einen Tropfen Augen Schmiermittel in jedes Auge und schließen Sie die Augen. </li><li> Stellen Sie das Heizkissen auf 36 ° C Grad zu Unterkühlung zu minimieren. </li><li> Prüfen Sie, ob das Fehlen von Fuß und Schwanz Reflexe durch Kneifen Sie die hinteren Pfoten und den Schwanz. </li></ol> 3. Chirurgie <ol><li> Reinigen Sie die Haut, die den Bauch mit 70% Ethanol. </li><li> Verwenden einer Pinzette, um die Haut nach oben ziehen. Nach jeder Operation, waschen alle Instrumente mit Reinigungsmittel und Wasser, und bei 72 ° CO / N sterilisiert. </li><li> Einen kleinen vertikalen Einschnitt (ca. 2 cm) entlang der Mittellinie ausgehend mittig zwischen der dritten und vierten Paare von Brustdrüsen. Verwenden Sie Fein Chirurgie Schere. Achten Sie auf die Bauchwand nicht beschädigen. </li><li> Trennen die Haut sanft von dem Bauchfell. </li><li> Visualisieren Sie die Arterienund Venen an der Bauchwand. Machen Sie eine weitere 2 cm vertikalen Schnitt durch die Bauchfell (Vermeidung der Gefäße), um die Bauchhöhle aus. </li><li> Klemmen Sie die Haut und Bauchwand auf jeder Seite die Vermeidung von Einklemmen der Arterien. Hinweis: Wenn die Arterien versehentlich punktiert, opfern das Tier sofort, indem Genickbruch oder Isofluran Überdosis. </li><li> Vor erwärmen die sterile PBS auf 37 ° C. Decken Sie die Klemmen mit PBS feucht Kim wischt. </li><li> Befeuchten die freiliegenden Bauchhöhle mit PBS. Wiederholen Sie diesen Schritt mehrmals während der Durchführung der Operation. </li><li> Verwendung von Rundzangen, ziehen Sie den embryonalen Kette (E15.5) weg von der Kavität. Lassen Sie die Kette auf den Rest Feuchttücher. Starten, indem die eine Hälfte der Gebärmutter zuerst. Sobald die Embryonen in ihr elektroporiert werden, bringen sie zurück nach innen, dann auf der zweiten Hälfte zu arbeiten. </li></ol> 4. Elektroporation <ol><li> Weiter Rundzange verwenden, um die Embryonen zu manipulieren. </li><li> Visualize die lateralen Ventrikel. Die lateralen Ventrikel entlang der Mittellinie (Abbildung 1b) laufen. </li><li> Hinzufügen Fast Green (Lager: 10% w / v) zu der DNA-Lösung 1:20 Mischung, um sie während der Injektion zu visualisieren. Bereiten Sie die DNA-Lösung durch Auflösen der aus einem Standard-Maxi-Prep-Reinigungsprotokoll in destilliertem Wasser erhaltenen Pellets. </li><li> Verwenden Sie vor gezogen Mikropipetten mit einem Stempel zu injizieren 1 ml DNA-Lösung (Dlx5 / 6-eGFP, 3 ug / ul) mit Fast Green. Schneiden Sie die Spitze der Mikropipette mit einer feinen Pinzette # 5 (Dumont) bis 6 mm Länge vom Anfang der Schulter bis zum Ende der Spitze der Nadel zu verlassen, bevor das Laden der Pipette. Halten Sie den Kopf des Embryos mit runden Pinzette. In das Gehirn mit der Pipette in einem 45 ° Winkel mit dem Ziel für den lateralen Ventrikel in der Nähe der Mittellinie angeordnet ist. Wenn die Nadel durch die Herzkammer dringt, langsam zurückziehen der Pipette, wie Sie den Kolben verwenden, um DNA zu injizieren. Der Ventrikel wird grün, wenn dieLösung beginnt, es zu füllen. An diesem Punkt nicht mehr bewegen die Pipette und spritzen 1 ml DNA. Sanft, ziehen Sie die Pipette. </li><li> Stellen Sie die CUY21 Elektroporator zu fünf 45V Impulse von 50 ms von 950 msec Abstand. </li><li> Verwenden Sie 5 mm Paddelelektroden für E15.5 Embryonen. Tauchen Sie die Elektroden in PBS, um eine effiziente Stromübertragung zu gewährleisten. </li><li> Platzieren Sie die Elektrode Paddel um den Kopf des Embryos mit der positiven Paddel auf der Herzkammer, die die DNA-Lösung. Leicht senken die positive Paddel mit Bezug auf die negative, eine ventrale Strom durch die Ganglien Eminenzen erstellen. Diese Manipulation wird ektopische Expression in Pyramidenzellen zu minimieren. </li><li> Nach der Platzierung der Elektroden, liefern einen Impuls, indem Sie das Fußpedal einmal. </li><li> Entfernen Sie die Elektroden und wischen Sie sie sauber. Wiederholen Sie die Schritte 4,6-4,9 mit jedem Embryo. </li><li> Nach Elektroporation alle Embryonen, pour 3 ml PBS in die Bauchhöhle und bringt sie in die Bauchhöhle. </li><li>Erste Naht der Bauchwand mit einer gebogenen Nadel und 5-0 Seidennaht und dann die Haut. Anwenden Lidocain (4%) auf die Wunde. Verwalten 120 mg / kg Aspirin inner Peritoneum zur Analgesie. Hinweis: Das gesamte Verfahren sollte in 20 min oder weniger durchgeführt werden. Es ist ratsam, dass Anfänger nur elektroporieren einige Embryonen, um die Betriebszeit kurz zu halten. Längerer Operationen wird die Überlebensrate der Embryonen zu verringern. </li><li> Nehmen Sie das Tier aus der Narkose Schläuche und eine Wiederherstellung auf dem Heizkissen auf einem Papier zu wischen. </li><li> Zurück Tier in den Käfig, halten Sie es auf dem Heizkissen bei 37 ° C und den Gesundheitszustand überwachen. Die Tiere in der Regel gut vertragen und die Operation beginnen sich langsam zu bewegen, kurz nachdem sie aus der Narkose Schlauch entfernt werden. Wenn übermäßige Blutungen oder Lethargie beobachtet wird, zu opfern das Tier sofort, indem IACUC genehmigt Euthanasie Verfahren wie Genickbruch oder Anästhesie Überdosis. </li><li> Zurück zu dem Käfig vivarium. Die Weibchen werden Geburt natürlich geben. </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>

Die Autoren haben nichts zu offenbaren.

Grenzen der Technik Während diese Technik ermöglicht Zelle autonom Analyse von Zellprozessen, ist es nicht geeignet für die Populationsanalyse. Die Elektroporationen sind sehr spärlich mit weniger als tausend Zellen elektroporiert pro Gehirn. Als Folge kann die Technik nicht verwendet werden, um Verhaltenskonsequenzen durch die genetische Manipulation von CGE abgeleiteten Inter bewerten. Während Elektroporationen durchgeführt bei E13.5 E14.5-Ziel MGE-Subtyp...

Der Großteil der kortikalen GABAergen Interneuronen stammen aus zwei transiente embryonale Strukturen nannte die medialen und Schwanzganglien Eminenzen (MGE und CGE jeweils) 4. Parvalbumin und Somatostatin exprimierenden Inter entstehen in der Erwägung, dass MGE Calretinin (Cr), Vasointestinal Peptid (VIP) und Reelin (Re) ausdrückt Inter stammen aus der CGE. Diese Intern Subtypen können durch ihre Geburtsdaten unterschieden werden. MGE abgeleitet Subtypen zwischen embryonalen Tag 9.5 (E9.5) und E16.5 5,6 geboren. Im Gegensatz dazu CGE abgeleitet Inter aus E12.5 E18.5 durch ihre Produktionshöchststand von 6 E15.5 geboren. Die genetische Targeting dieser spät geboren Bevölkerung bleibt jedoch unklar. Die murinen Distal-less (Dlx) Gene werden ausschließlich in den Entwicklungs ventralen Vorderhirn 3 ausgedrückt. GABAergen Interneuronen und Projektionsneuronen des Striatums, aber nicht Pyramidenzellen der Hirnrinde auszudrücken DLX1, 2,5, und 6 Gene in frühen Entwicklungsstadien 3. Tatsächlich sind die Deluxe, Gene in der MGE und CGE Subventrikularzone (SVZ) in allen GABAergen Vorläuferzellen exprimiert wird. Expression dieser Gene wird eingeschränkt, um Subtypen bei postmitotische Stufen 7-9 wählen. Vorherige experimentelle Beweismaterial zeigte, daß das Dlx5 / 6 Enhancer-Element ermöglicht die selektive Ausrichtung der GABAergen Linien in transgenen Mausmodellen 2. Wir haben die Verwendung von einer dieser Enhancer-Elementen im Rahmen der episomalen Expression in sich entwickelnden Maus-Gehirn. Wir Teil zusammen mit einem Minimalpromotor und dem enhanced green fluorescent protein (eGFP) in einen Bluescript (BS) Backbone-Plasmid (Figur 1) kloniert den Dlx5 / 6-Enhancer-Element. Wir haben das Plasmid durch Elektroporation in utero bei E15.5 selektiv auf Cr-, VIP-und Research-Subtypen 3,8,10. Unsere Technik ermöglicht spärlich Elektroporation, die erleichtertdie Rekonstruktion der morphologischen Merkmale singe Zellen. Darüber hinaus ist die außerordentlich hohe Genexpression in kortikalen GABAergen Neuronen ermöglicht funktionelle Studien. Wir führten Verlust und Gewinn von Funktionsprüfungen mit mehreren Wildtyp und dominant negative Gene 11.

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subtype-selective electroporation of cortical interneurons

Die Studie des Zentralnervensystems (ZNS) Reifung beruht auf genetischer Ausrichtung der neuronalen Populationen. Allerdings hat die Aufgabe, die Beschränkung der Expression von Genen von Interesse spezifischen neuronalen Subtypen bemerkensanspruchs aufgrund der relativen Knappheit von spezifischen Promotorelemente bewährt. GABAergen Interneuronen bilden einen neuronalen Population mit umfangreichen genetischen und morphologischen Vielfalt. Tatsächlich haben mehr als 11 verschiedene Subtypen von GABAergen Interneuronen in der Maus Kortex 1 gekennzeichnet. Hier präsentieren wir ein angepasstes Protokoll für selektive Ausrichtung der GABAergen Populationen. Wir erreicht Subtyp selektive Ausrichtung von GABAergen Interneuronen mit Hilfe der Enhancer-Element des Homeobox Transkriptionsfaktoren Dlx5 und Dlx6, Homologen des Drosophila-Distal-less (DLL)-Gen 2,3, um die Expression spezifischer Gene durch in utero Elektroporation zu fahren.

Wir passten die in utero Elektroporation Technik, um Zelltyp-spezifische Ausrichtung der Reifung Neuronen zu erreichen. Um die Expression von eGFP in CGE-abgeleitete Inter zu fahren, haben wir die Dlx5 / 6 Enhancer-Element und beschränkt unsere Injektionen E15.5, die Bühne, wenn die Mehrheit der CGE-abgeleitete Inter erzeugt. Wir führten die Analyse auf P8 und P15 11 (Abbildungen 1 und 2). Wir bestätigten die ventrale Ursprung elektroporiert Neuronen dur...

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.

Subtyp-selektive Elektroporation kortikaler Inter

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 Aufrufe.  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.

Serie: 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 Elektroporation gefolgt von primären neuronalen Kultur für ein Studium Gene Function in Subset von kortikalen Neuronen

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

Forschung

Neurowissenschaften

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.

In-vivo-Elektroporation von Entwicklung Maus Retina

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.

Isolation und Kultur von Maus Kortikale Astrozyten

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

Neonatal Subventrikulärzone Elektroporation

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 Elektroporation und Whole Hemisphäre Explantate: A Simple Experimentelle Methode für Studien der frühen kortikalen Entwicklung

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.

Neonatal Pial Oberflächen Elektroporation

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.

Whole-Cell-Patch-Clamp-Aufnahmen aus Morphologically- und neurochemisch identifizierten Hippocampus Inter

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.

Ein Leitfaden zur<em&gt; In vivo</em&gt; Single-Unit-Aufnahme von Optogenetically Identifizierte kortikalen inhibitorischen Inter

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.

Elektrophysiologische Verfahren zur Aufzeichnung von intrazellulärer Spannung Antworten von<em&gt; Drosophila</em&gt; Fotorezeptoren und Inter auf Lichtreize<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.

Visualisierung der kortikalen Module in abgeflachten Säugetier-Rinde

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