Wir danken Dr. Takahiko Matsuda für die Bereitstellung der pCAG-mCherry konstruieren. YCM wird durch einen Career Development Award der Schweppe-Stiftung und ein Stipendium der Whitehall-Stiftung unterstützt.

1. Versorgung Vorbereitung (nicht im Video) <ol><li> Bereiten Sie einen frischen 1X Verdünnung von Penicillin / Streptomycin (Pen / Strep) in 1X PBS. Nicht mehr als 50 ml notwendig sind. Filter mit einer Spritze 0,22 um-Filter. </li><li> Bereiten Injektion Konstrukte durch die Kombination von gewünschten Plasmid-Konstrukte. Wir haben alle Konstrukte in der pCAG-Flag-Vektor mit geeigneter Stöchiometrie bis zu einem Endvolumen von ~ 10 l. Außerdem sollte pCAG-GFP 14 oder pCAG-mCherry zum Verfolgen Elektroporation Effizienz und elektroporierten Zellen gegeben werden. Wir verwenden pCAG-mCherry für dieses Experiment. Schließlich werden 0,22 um filtriert Fast Green-Farbstoff in der Plasmid-DNA Mischung bei einer Endkonzentration von 0,05%, um die Einkoppeleffizienz in Embryonenküken midbrains visualisieren. </li></ol> 2. Egg Vorbereitung (im Video) <ol><li> Um erwerben HH Stadium 11 Embryonen, brauchen Eier bebrütet werden bei 100 ° F (37,8 ° C) für etwa 41 Stunden nach der Befruchtung mit 25-40% Luftfeuchtigkeit. OrtEier auf ihrer Seite und markieren die Spitze jeder Strich mit einem Bleistift. Platzieren Sie Trays in einer vorgewärmten, befeuchteten Inkubator. Langsam Eier schaukeln, indem Sie die Drehfunktion des Inkubator-Plattformen auf &quot;Automatisch&quot;. Überprüfen Sie die Feuchtigkeit und Wasserstand alle 24 Stunden. </li><li> Kurz vor Beginn des Versuchs, zu entfernen Eier aus dem Inkubator verlassen und bei Raumtemperatur. Dies wird dazu beitragen, die Entwicklung verlangsamen und verhindern, dass über die Entwicklung. </li><li> Reinigen Sie die Oberfläche der einzelnen Eier mit 70% Ethanol und wischen Sie mit einem Kimwipe. </li><li> Nehmen Sie zwei 4 cm Stücke von Klebeband und kleben Sie diese Seite an Seite auf dem Ei, ein großes Rechteck über den Bleistift Strich zu machen. Glätten Sie das Band vor dem Ei. Dies ist, wo das Fenster gemacht werden. Das Band wird kleine Stücke von Schalen abfällt, wenn das Fenster aufgeschnitten zu verhindern. </li><li> Verwenden Sie eine 10 ml-Spritze und 18-Gauge-Nadel ½ bis 5 ml Albumin aus dem Ei zu ziehen. Nach dem Durchstechen der Schale, winkeln Sie die Nadel weg von der Dotter nicht zu stören, umoder Schäden am Embryo beim Entfernen Albumin. Reinigen Sie die Nadel mit 70% Ethanol zwischen Eiern, aber sicher sein, die Nadel ist vor Durchstechen der nächste Ei trocken. </li></ol> 3. Glass Injektionsnadel Vorbereitung (im Video) <ol><li> Injektionsnadeln sind gemacht mit den folgenden Einstellungen auf einem lodernden / Braun Feinpipettenziehvorrichtung (Sutter Instrument Modell P-97): Heat 670, Pull-120, Vel 40, 165 Zeit. World Precision Instruments (API) 4 Zoll 1,0 mm dünnen Wand Kapillargläser (API TW100F-4) wird verwendet, um Nadeln zu machen. </li><li> Laden Sie ein Glas gezogen Nadel mit einer P10-Pipette und die lange Spitze Kapillare Laden Pipettenspitze (Eppendorf 5242956,003). Legen 5-10 ul, je nachdem wie viele Eier sind, die injiziert werden. Füllen Sie das Glas langsam Nadel, die Schaffung eines durchgehenden Spalte von Plasmid-Lösung abwesend von Luftblasen. </li><li> Platzieren Sie die geladene Nadel in den Nadelhalter an dem Mikromanipulator (Narishige MM3) und dem Picospritzer III Mikroinjektorkopfs. Sehen Sie sich die Nadel unter einem dissectiMikroskop auf und schneiden Sie die Nadel an der Stelle, wo die grüne Flüssigkeit stoppt. Es hilft, einen dunklen Hintergrund zu verwenden, um besser zu visualisieren Sie die Nadel. Die richtige Nadel Trimmen kommt mit der Praxis. </li></ol> 4. Mittelhirn / Neuralrohr Injection (im Video) <ol><li> Verwenden Sie eine Schere Frühjahr 2-2,5 cm Durchmesser Fenster im Ei geschnitten. Starten Sie das Fenster aus dem vorigen Stichloch machte beim Zeichnen von Albumin, und drehen Sie das Ei in der Hand, wie Sie abgeschnitten. Seien Sie sicher zu reinigen Sie die Schere mit einem Kimwipe in 70% Ethanol zwischen Eiern gesättigt. </li><li> Alle Hühnerembryonen unter einem Dissektionsmikroskop. Es kann notwendig sein, um die Augen zu trainieren. Es kann helfen, den Embryo durch Einspritzen von 0,22 um-gefiltert 20% Tusche in PBS unter dem Hühnerembryo zu visualisieren. Wir haben jedoch gefunden, dass dieser Schritt vermieden werden sollte, wenn möglich, wie es für das Überleben tendenziell abnimmt. </li><li> Injektion funktioniert am besten, wenn der Embryo in parallel zu dem Weg der Nadel angeordnet ist, wobei der Kopf von der Nadel.Langsam dringen das Neuralrohr in einem 45 Grad Winkel zum Mittelhirn-Hinterhirn Kreuzung. Es ist sehr wichtig, nur füllen die neuronale Vesikel, die den Mittelhirn mit Plasmid-DNA und Fast Green Farbstoff, ohne nennenswerte Diffusion in die Vorderhirn und Hinterhirn Regionen. Starten Sie mit der Injektion von Picospritzer Mikroinjektor die Dauer Satz bei 25 ms, das eventuell verändert je nach Nadel Trimmen werden. In der Regel zwischen 10-50 ms verwenden. </li></ol> 5. Elektroporation (im Video) <ol><li> Rufen Sie die Injektionsnadel. Platz 3 Tropfen 1X Pen / Strep in PBS-Lösung in das Ei auf den Embryo. </li><li> Überprüfen Sie die Einstellungen des BTX EMC830 Elektroporator: <table border="1"><tbody><tr><td> SPANNUNG: </td><td> 20 V </td></tr><tr><td> PULSLÄNGE: </td><td> 25 ms </td></tr><tr><td> # Impulse: </td><td> 3 </td></tr><tr><td> INTERVAL: </td><td> 500 ms </td></tr><tr><td> POLARITÄT: </td><td&gt; UNIPOLAR </td></tr></tbody></table></li><li> Die 2 mm langen L-förmigen Platinelektroden auf der gleichen horizontalen Niveau wie der Boden des Neuralrohrs, mit der embryonalen Mittelhirn sitzt in der Mitte zwischen zwei beabstandeten Elektroden 3 mm. Ausrichten der Elektroden mit dem Boden des Neuralrohrs ist entscheidend für eine effiziente Elektroporation des ventralen Mittelhirn. Um Mittelhirn-spezifische Elektroporation zu erreichen, ersetzten wir die Original-BTX-Elektroden mit zwei maßgeschneiderten Platin L-förmigen Elektroden mit kurzen 2 mm langen Spitzen. Diese Platin-Elektroden von 0,5 mm Durchmesser Platindrähte (Alfa Aesar) hergestellt. Diese Platin-Elektroden sind gerade lang genug, um das Mittelhirn eines Stufe 11 Hühnerembryonen, die etwa 0,9 mm lang, ohne Targeting viel von dem Vorderhirn oder den Rautenhirn Regionen ist zu decken. Drücken Sie die BTX-Fußschalter einmal. Luftblasen um die Elektroden mit jedem erfolgreichen Elektroporation erzeugt werden. Entfernen Sie vorsichtig die Elektroden aus dem Ei und wischen Sie die elektrOden mit einem 70% igen Ethanol-Kimwipe abgedeckt. Setzen Sie noch 3 Tropfen 1X Pen / Strep-Lösung im Ei, auf den Embryo. </li><li> Decken Sie das Fenster mit einem 5 cm x 5 cm großes Stück klar Packband. Achten Sie darauf, um das Ei gut und zu versiegeln, dass die Ei-Inhalte nicht in Kontakt mit der Band zu kommen. </li></ol> 6. Inkubieren und Ernte <ol><li> Legen Sie die Eier wieder in den Inkubator. Stellen Sie sicher, das Drehen des Inkubator-Plattformen ist AUS. Inkubieren Eier, bis die gewünschte HH 23 wird erreicht. </li><li> Ernte lebende Embryonen und sie in gemeinsamen PBS gefüllten Petrischalen für jede Bedingung. Bildschirm geerntet Embryonen unter einem Fluoreszenz-Mikroskop Dissektion und behielt nur Embryonen mit mCherry Ausdruck. </li><li> Übertragen Embryonen in einzelne Vertiefungen einer 24-Well-Platte. Füllen Sie jede gut mit 1 ml frisch zubereiteten 2% Paraformaldehyd (PFA) und lassen Sie sie 2-3 Stunden bei 4 ° C fixieren </li><li> Waschen fixierte Embryonen dreimal für jeweils 10 Minuten in PBS. Ort Embryonen nacheinander in 15% und 30% Saccharose, bis ins Gleichgewicht gebracht. Die Embryonen werden zunächst in Saccharose zu schweben, wird aber beim Äquilibrierung sinken. </li><li> Überprüfen Embryonen unter einem Fluoreszenz-Mikroskop Dissektion, sich Notizen auf dem mCherry Expressionsmuster und Niveau für jeden Embryo. Einbetten jedes Embryo im Oktober Es ist sehr kritisch zu orientieren Embryonen richtig optimale Mittelhirn Querschnitte für nachfolgende Analysen (1) zu erzeugen. </li><li> Bereiten 18 um dicken Mittelhirn Abschnitten mit einem Leica CM3050S Kryostaten. Analysieren Mittelhirn Entwicklung und Differenzierung von dopaminergen Neuronen Immunfärbung mit geeigneten Zelltyp Marker. </li></ol> 7. Repräsentative Ergebnisse Eine schematische Darstellung der Elektroporation und Analyse Embryonenküken Mittelhirn ist in Abbildung 1 dargestellt. In den erfolgreich injiziert und elektroporiert Hühnerembryonen, sollte Ausdruck sein mCherry im Mittelhirn Region sichtbar nach weiteren Entwicklung ( <strong&gt; 2A, B, C). Unter der Annahme, dass mCherry mit der Expression anderer Gene co-elektroporierte korreliert, können Embryonen ausstellenden richtige Ausdruck mCherry dann fixiert und eingebettet werden, um Mittelhirn Kryoschnitten für weitere Untersuchungen (2D) vorzubereiten. Im Allgemeinen kann etwa 50% der ventralen Mittelhirn neuralen Vorläuferzellen effizient mit dem obigen Protokoll transfiziert werden. Die Spezifikation der dopaminergen Schicksal in elektroporierte neuralen Vorläuferzellen durch die Co-Lokalisation von mCherry untersucht werden kann, Gen-Flag-markierte, mit dopaminergen Neuronen-Marker wie Tyrosin-Hydroxylase (TH) (Abbildung 3). Die Strukturierung der Vorläuferzellen Domänen in dem ventralen Mittelhirn kann durch Co-immnostaining Gefrierschnitte mit unterschiedlichen Vorläuferdomäne Marker untersucht werden. Zu wenig mCherry Ausdruck legt nahe, dass ektopische Gene wahrscheinlich nicht effizient ausgedrückt. Embryonen unter dieser Bedingung nicht für weitere Untersuchungen verwendet werden. Ebenso, dass Embryonennicht bis zum Ende des experimentellen Verfahrens überleben können nicht für die Datensammlung und Analyse verwendet werden. <img alt="1" src="/files/ftp_upload/4017/4017fig1.jpg" /> Abbildung 1. Illustration der Embryonenküken Mittelhirn in ovo Elektroporation Assay. HH Stadium 11 embryonalen Hühnerembryonen mit pCAG-mCherry zusammen mit Genen von Interesse mit schnellen Grün gemischt, um die Einspritzung zu visualisieren (A) injiziert. Nach mit DNA-Konstrukten gefüllt sind, midbrains mit einer Rechteckwelle Elektroporator elektroporiert. Die Embryonen werden weiter inkubiert, bis die gewünschte HH Stufe 23 erreicht ist. Dann mCherry-positiven Embryonen werden geerntet, fixiert und Embedded (B). Mittelhirn Kryoschnitten sind mit Schnittebenen durch die weiße Linie in Abbildung 1B angegeben vorbereitet und analysiert durch Immunfärbung mit Mittelhirn Marker wie Tyrosin-Hydroxylase (TH) (C). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank">Klicken Sie hier</a>für eine größere Abbildung zu sehen. <img alt="2" src="/files/ftp_upload/4017/4017fig2.jpg" /> Abbildung 2. Vorbereitung der Gefrierschnitte aus Embryonenküken Mittelhirn. Nach 41 Stunden Inkubation werden HH Stadium 23 Hühnerembryonen auszudrücken mCherry und Gene von Interesse geerntet (A, B und C), mit PFA fixiert, behandelt mit Saccharose, und eingebettet in Oktober für Kryoschneiden. Hühnerembryonen werden sorgfältig ausgerichtet, um die Vorbereitung des Mittelhirns Abschnitten zu gewährleisten. Eine typische Embryo Morphologie und die Schnittebene für den Erhalt von Mittelhirn Abschnitte werden gezeigt (D). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> . <img alt="Abbildung 3" src="/files/ftp_upload/4017/4017fig3.jpg" /> Abbildung 3. Analyse der elektroporierte Mittelhirn Abschnitten. Gefrierschnitte von Embryonen mit hoher mCherry Ausdruck (A) werden hergestellt und mit Antikör gefärbten, die die Flag-markierte Gen von Interesse (B) und dopaminergen Mittelhirn Neuronenmarkers TH (C) zu erkennen. Embryonale midbrains mit pCAG-mCherry und pCAG-Flag leeren Vektor als Kontrolle dienen elektroporiert. <a href="http://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> .

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Die Autoren haben keinerlei potenzielle Interessenkonflikte offen zu legen.

Die in-ovo-Elektroporation von Embryonenküken Mittelhirn bietet eine kostengünstige und schnelle Alternative zur Erzeugung von transgenen oder Knockout-Tieren in vivo-Studie von Genfunktionen in dopaminergen Mittelhirn-Neuronen Entwicklung durchzuführen. Verwendet man die 2 mm langen L-förmigen Platinelektroden mit embryonalen Mittelhirn-spezifischen DNA-Injektion ist der Schlüssel für eine effiziente Expression des Gens von Interesse in dopaminergen Mittelhirn-Neuronen Vorläuferzellen. Darüber...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Befruchtete Hühnereier </td><td> Charles River Laboratories </td><td></td><td></td></tr><tr><td> Egg Brutkasten </td><td> DQR Fertigung </td><td> 1502 Sportsman </td><td></td></tr><tr><td> BTX Electroporator </td><td> BTX Harvard Apparatus </td><td> ECM830 </td><td></td></tr><tr><td> Elektroden </td><td> BTX Harvard Apparatus </td><td> 45-0162 L-förmigen genetrodes </td><td> Für den Einsatz mit ECM830 Elektroporator </td></tr><tr><td> Platin-Iridium-Draht </td><td> Alfa Aesar </td><td> # 10056 </td><td> 0,5 mm Durchmesser Zur Herstellung der 2 mm langen Elektroden </td></tr><tr><td> Glaskapillare </td><td> World Precision Instruments </td><td> TW100F-4 </td><td></td></tr><tr><td> Microloader Pipettenspitze </td><td> Eppendorf </td><td> 5242 956.003 </td><td></td></tr><tr><td> Tusche </td><td> Staples </td><td></td><td> Filter vor der Verwendung 20% </td></tr><tr><td> Mikroinjektor </td><td> Parker Automation, Parker Hannifin Cooperation </td><td> Picospritzer III </td><td></td></tr><tr><td> Fluoreszierende Dissektionsmikroskop </td><td> Leica </td><td> MZ16F </td><td></td></tr><tr><td> Feinpipettenziehvorrichtung </td><td> Sutter Instrument </td><td> D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Dopaminergen Neuronen in der ventralen Mittelhirn Kontrolle Bewegung befindet, emotionales Verhalten und Belohnung Mechanismen 3.1. Die Dysfunktion des ventralen Mittelhirn dopaminergen Neuronen in der Parkinson-Krankheit, Schizophrenie, Depression und Demenz 5.1 verwickelt. So konnte die Untersuchung der Regulation der dopaminergen Mittelhirn-Neuronen Differenzierung nicht nur liefern wichtige Einblicke in die Mechanismen, die Mittelhirn Entwicklung und neuronale Vorläuferzellen Schicksal Spezifikation, sondern auch zur Entwicklung neuer therapeutischer Strategien zur Behandlung einer Vielzahl von humanen neurologischen Erkrankungen. Dopaminergen Neuronen differenzieren aus neuralen Vorläuferzellen entlang der ventrikulären Zone der embryonalen ventralen Mittelhirn. Die Entwicklung von neuralen Vorläuferzellen wird durch Genexpression 6,7 gesteuert. Hier berichten wir über Techniken der Verwendung von Elektroporation, um Gene gezielt zum Ausdruck bringen im Mittelhirn der Hamburger Hamilton (HH) Stufe 11 (thirteen Somiten, 42 Stunden) Hühnerembryonen 8,9. Die externe Entwicklung von Hühnerembryonen ermöglicht die komfortable experimentelle Manipulationen an bestimmten embryonalen Stadien, mit den Auswirkungen auf späteren Entwicklungsstadien Zeitpunkten 10-13 bestimmt. Küken embryonalen neuralen Röhren früher als HH Stadium 13 (neunzehn Somiten, 48 Stunden) bestehen aus multipotenten neuralen Vorläuferzellen zur Differenzierung in verschiedene Zelltypen des Nervensystems sind. Die pCAG Vektor, der sowohl einen CMV-Promotor und ein Küken β-Actin-Enhancer enthält, kann für robuste Expression des Flag oder andere Epitop-markierten Konstrukte in Embryonenküken neuronalen Röhren 14. In diesem Bericht möchten wir betonen, besondere Maßnahmen zur regional begrenzten Gen-Expression in embryonalen dopaminergen Mittelhirn-Neuronen-Vorläuferzellen, einschließlich wie man injizieren DNA-Konstrukte gezielt in den embryonalen Mittelhirns Gegend und wie Elektroporation mit kleinen maßgeschneiderten Elektroden lokalisieren zu erreichen. Analysieren von chick Mittelhirn in späteren Stadien eine hervorragende in vivo für Plasmid-Vektor-vermittelte gain-of-Funktion und Loss-of-Funktions-Untersuchungen der Mittelhirn Entwicklung. Modifizierung des experimentellen System kann des Assays, anderen Teilen des Nervensystems erstrecken zum Durchführen Gastrula-Analyse und zur Untersuchung der Regulation der Genexpression.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Um die Funktion und die Regulation von Genen während der Entwicklung der dopaminergen Neurone im Mittelhirn zu bewerten, beschreiben wir eine Methode, die beinhaltet<em&gt; In-ovo-</em&gt; Elektroporation von Plasmid-DNA-Konstrukte in embryonale Küken ventralen Mittelhirn dopaminergen Neuronen Vorfahren. Diese Technik kann verwendet werden, um eine effiziente Expression von Genen des Interesses, um verschiedene Aspekte des Mittelhirns Entwicklung und Differenzierung dopaminergen Neuronen zu untersuchen.

In ovo Elektroporation in Mittelhirn-Küken für Studien über Genfunktion in dopaminergen Neuronen Entwicklung

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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Neuroscience

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

11.3K Aufrufe.  Children's Hospital of Chicago Research Center. Um die Funktion und die Regulation von Genen während der Entwicklung der dopaminergen Neurone im Mittelhirn zu bewerten, beschreiben wir eine Methode, die beinhaltet In-ovo- Elektroporation von Plasmid-DNA-Konstrukte in embryonale Küken ventralen Mittelhirn dopaminergen Neuronen Vorfahren. Diese Technik kann verwendet werden, um eine effiziente Expression von Genen des Interesses, um verschiedene Aspekte des Mittelhirns Entwicklung und Differenzierung dop...

Serie: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

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 ...

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Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Die Anwendung eines NMDA-Rezeptor-Leitwert in Rat Mittelhirn dopaminergen Neuronen Mit der Dynamic-Clamp-Technik

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Die Transfektion von Maus retinalen Ganglienzellen durch In vivo Elektroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Maus in Utero Elektroporation: Kontrollierte Spatiotemporal Gene Transefection

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

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

In ovo Expression von microRNA in ventralen Mittelhirn-Küken

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Regie dopaminergen Neuron Differenzierung von humanen pluripotenten Stammzellen

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

In Vivo Calcium Imaging der Seitenlinie Haarzellen im Larvenstadium Zebrafisch

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Studium der Funktion des Gehirns bei Kindern mit Magnetoenzephalographie

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

3D Kinematische Analyse zur funktionellen Bewertung im Rattenmodell von Ischias-Nerven-Crush-Verletzungen

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...