This work is supported by a National Science Foundation award (1145581) to LWG.

HINWEIS: Das gesamte Verfahren wurde in Übereinstimmung mit den Richtlinien der National Institutes of Health durchgeführt, wie von der Animal Care und Verwenden Ausschuss der Universität von Illinois in Chicago genehmigt. 1. Lösungen und Kultur Medien Vorbereitungen <ol><li> Halten Sie alle Lösungen bei -20 ° C für bis zu sechs Monate. Halten Sie das Kulturmedium bei 4 ° C für bis zu 3 Monate. </li><li> Planen 100 ml Kulturmedium durch Mischen von 1 ml Pen-Strep-Lösung und 1 ml itsX (Insulin-Transferrin-Selen-Ergänzung) auf 100 ml mit DMEM. </li><li> Vorbereitung Enzymlösung durch Mischen mit 250 ml DMEM, 2,5 ml 100 mM CaCl 2 und 2,5 ml 50 mM EDTA. </li><li> Vorbereitung Inaktivierung durch Mischen von 225 ml DMEM, 25 ml FBS, 625 mg Albumin und 625 mg Trypsininhibitor. </li><li> Bereiten Sie eine Locke-Lösung von 154 mM NaCl, 5,6 mM KCl, 5,0 mM HEPES, 3,6 mM NaHCO3, 5,6 mM Glucose. Stellen Sie den pH-Wert mit NaOH auf 7,3. </li><li>Vorbereiten einer Poly-D-Lysin (PDL) Lösung von 50 mg / ml. Verwenden eine Endkonzentration von 1 mg / ml beschichtet Deck. </li><li> Vorbereiten eines extrazellulären Lösung von 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2, 10 mM HEPES-NaOH und 10 mM Glucose. Den pH-Wert auf 7,3 mit NaOH mit einer Osmolarität von ~ 310 nmol / kg. </li><li> Vorbereiten einer Pipettenlösung von 50 mM NaCl, 100 mM TEACl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2 und 10 mM HEPES. PH auf 7,3 mit NaOH mit einer Osmolarität von ca. 290 nmol / kg. </li></ol> 2. Nebenniere Isolation <ol><li> Autoklaven ein Paar von großen und kleinen Dissektion Schere und zwei Paar Pinzette. Schutzhandschuhe tragen während des gesamten Prozesses und setzen Sie den Seziersaal Tablett auf einem Eimer Eis. </li><li> Verwenden Mäuse an Tag 0-3 übernommen. Tiere einschläfern mit einem Drucktank CO 2, gefolgt von Enthauptung. </li><li> Nutzen die kleineren Satz von Schere, um die Rückseite der Welpen nach der Wirbelsäule aus Cervi reduzieren<html> cal zu Region kaudal. Achten Sie darauf, oberflächlich unter der Haut geschnitten und nicht in die Körperhöhle zu durchbohren. </li><li> Als nächstes Haut weg von der Wirbelsäule, so dass Muskulatur ausgesetzt ist, um eine klare Sicht auf die Wirbelsäule haben. Von hier aus, machen Sie einen Schnitt an der transectional zervikalen Rückenmark und dann zwei parallele Schnitte an den Seiten der Wirbelsäule. </li><li> Mit einer Zange hält den Hauptkörper des Tieres, mit der anderen Pinzette zu greifen und Wirbelsäule abziehen Wirbelsäule bis Nieren ausgesetzt sind. </li><li> In diesem Moment sichtbar die Nebennieren an der Oberseite der Nieren. Dann vorsichtig die zwei Drüsen von jedem Tier zu einem konischen Rohr mit Locke-Lösung gefüllt. HINWEIS: Manchmal sind die Drüsen auf der Zange festhalten. Wenn dies der Fall ist, leicht zu verwenden weiteres Paar von Zangen, um die Entfernung der Drüsen aus der Zange und in die Locke-Lösung zu unterstützen. Die Drüsen können in diesem Zustand für bis zu 2 Stunden gehalten werden. </li></ol>e &quot;&gt; 3. Tissue Verdauung <ol><li> In der Zwischenzeit Blasen die Enzymlösung mit 5% CO 2 + 95% O 2 für 15 Minuten vor der Verwendung. Stellen Sie sicher, dass die Lösung ins Gleichgewicht dreht sich von einem hellen rosa Farbe in rosa / orange Farbe. </li><li> Füge das Papain auf die frisch geperlt Enzymlösung in einer Endkonzentration von 20-25 Einheiten / ml. Jedes Paar von Drüsen aus jedem Tier inkubieren 40-60 min bei 37 ° C in der Enzymlösung mit Papain. </li><li> Fügen 75% Inaktivierung Lösung in jedes Röhrchen und Inkubation bei 37 ° C für weitere 10 min. Diese Inkubation inaktiviert das Enzym-Aktivität von Papain. </li><li> Nach der 10 min Inkubation mit der Inaktivierung Lösung, übertragen Sie leicht die Drüsen in ein neu beschriftet Rohr und dann waschen die Drüsen 3x mit Kulturmedien. </li></ol> 4. Zelldissoziation und Oberflächen <ol><li> Nach dem Waschen, legen Sie die zwei Drüsen in 200 ul der Kulturmedien und dann sanft titriert die Drüsen undnach unten durch die Pipettenspitze mit einer 200 ul-Pipette, bis es nicht länger ein großes Stück des Gewebes in die Lösung. HINWEIS: Bei der Titration, zu erhalten Verschraubungen am Boden des Röhrchens mit einer Abwärtskraft von 200 ul Pipettenspitze und greifen das Gewebe vorsichtig und langsam. Seien Sie sicher, langsame, kontinuierliche Schläge zu verwenden, nie Pipettieren schnell oder abrupt. </li><li> Fügen Sie eine zusätzliche 250 ul Kulturmedien, um das Gesamtvolumen von Kulturmedien bis zu 450 ul zu bringen. </li><li> Platte 60 ul dieser Zellen enthaltenden Lösung auf jeder der sieben 12 mm PDL-pre-beschichtete Deckgläser, die in einer 60 x 15-mm-Kulturschale gegeben werden. </li><li> Einmal überzogen, die Schale in den Brutschrank, die bis 37 ° C mit einem stetigen Fluss / Versorgung von 5% CO 2 für 30 min eingestellt und gewartet werden, um eine Umgebung für die Lebensfähigkeit der Zellen zu gewährleisten. Nach der Inkubation vorsichtig mit 5 ml vorgewärmten Kulturmedien in der Kulturschale und gibt das Gericht zurück in den Brutschrank für 24 Stunden vor der Zell-Attached-Aufnahmen. HINWEIS: In der Regel können die Zellen für elektrophysiologische Aufzeichnungen für 4 Tage verwendet werden. </li></ol> 5. Zellidentifikation und Gigaohm Seal Bildung <ol><li> Legen Sie die 12 mm Deckglas in der extrazellulären Lösung. Da die ganze Nebennieren sind für die Zellherstellung verwendet, mischen die chromaffinen Zellen (in der Regel sehr hell mit einem bräunlichen / beige Farbgebung und einer fast perfekten Kreisform (Abbildung 1) in Kultur mit Rindenzellen (kleiner und dunkler als chromaffinen Zellen). </li><li> Ziehen Sie die Patch-Pipetten in vier Stufen unter Verwendung eines programmierbaren P-97 Magnet. Tauchen Sie die Pipettenspitze in einem geschmolzenen Wachs, die dazu beitragen, die Kapazität des Glas reduzieren und dann wird Feuer Lack die Spitze zu &quot;Blow-weg&quot; das Wachs aus dem Inneren der Pipettenspitze. HINWEIS: Bei der Patch-Pipette mit Lösung gefüllt, haben Patch-Pipetten typischerweise einen Widerstand von ~ 2 MOhm. </li><li> Nähern Sie sich dem Patch-Pipette in der Nähe der Zelle in mehreren microns. Um einen Cell-Attached-Konfiguration zu erreichen, suchen Sie nach jedem leichten Zell Verformung während der Annäherung der Patch-Pipette näher an der Zelle. Gelten, bis eine sanfte Saugwirkung GOhm Dichtung erreicht wird. </li></ol> 6. Cell-Attached-Kapazität Recordings <ol><li> Sobald ein GOhm Dichtung offenbart, machen Cell-Attached-Aufnahmen durch den Einsatz von einem EPC-7 sowie Patch-Clamp-Verstärker und ein Zwei-Phasen-Analog Lock-in-Verstärker (siehe beigefügte Tabelle der Ausrüstung). Anwenden eines Sinuswelle mit einer Amplitude von 50 mV (RMS) und einer Frequenz von 20 kHz von dem Lock-in-Verstärker. </li><li> Den Ausgangsfilter des Lock-in-Verstärker auf 1 ms Zeitkonstante und 24 db. Stellen Sie die Phase des Lock-in-Verstärkers, so daß transiente durch vorsichtiges Absaugen Impulse erzeugt Kapazitätsänderungen nur in der Patch-Kapazität (Im) Spur ohne Projektion in die Patch Leitfähigkeit (Re) Spur auf (Abbildung 2). HINWEIS: Bitte beachten Sie die Angaben für die Kalibrierung des Systems in reKonferenz 19. </li><li> Identifizieren typische Kapazitätsänderungen durch &quot;nach unten&quot; Schritte (Abbildung 3). HINWEIS: Eine solide Aufnahme wird als eine &quot;Treppe&quot; Art Ähnlichkeit mit vielen nach unten Schritte, die Internalisierung einzelne Vesikel zeigt an, betrachtet werden. Der Patch-Prozess dient als mechanische Stimulation seit Aktionsstrom kann in der Regel von den meisten Zellen aufgenommen werden. </li><li> Benennen Sie die Dateien als einzelne Ordner mit Datum: mm / tt / Jahr und jedes Cell-Attached-Aufnahme als seine eigene individuelle Nummerierung (xx) in dieser Datei. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Cell-Attached-Kapazitätsmessungen erfordern mehrere wichtige Schritte, um Aufnahmen mit hoher Qualität erfolgreich zu erhalten: 1) lebensfähig und gesunden Zellen von Nebennieren hergestellt werden; 2) PDL Beschichtung der Deck; 3) Gigaohm Dichtung Bildung; 4) Geräuschpegel des Systems; und 5) Phasenkorrektur. Für Tierchirurgie, ist eine potenziell Änderungen machen können, um den chirurgischen Ansatz, um am besten anpassen und Geschicklichkeit, um Schäden bei der Präparation zu ver...

Synaptischen Übertragung wird durch Exocytose von Neurotransmitter-haltigen synaptischer Vesikel vermittelten, und diese Vesikel müssen lokale endozytischen Recycling innerhalb der Nervenendigung unterziehen, um neuronale Kommunikation langfristig aufrecht zu erhalten. Angesichts der wesentlichen Rolle der synaptischen Übertragung im Gehirn, das Verständnis der molekularen Maschinerie, die synaptischen Vesikel-Zyklus ist eine wesentliche Grundlage für ein besseres Verständnis der zellulären Kommunikation als Ganzes darstellt. Unter Zellmodellsysteme, hat die chromaffinen Nebennierenzell einige der endgültige Einblick in die molekulare Maschinerie zugrunde liegenden synaptischen Vesikel-Recycling zur Verfügung gestellt. Exozytose, der letzte Schritt in der Neurotransmitterfreisetzung, wurde immens sucht und durch die Verwendung des chromaffinen Nebennierenzellen 1,2 untersucht. In der Tat sind die meisten der molekularen Spieler, die die Bildung, Ausrichtung, Docking und Fusion von sekretorischen Granula instrumentieren wurden aufgrund der Anwendung von div identifizierterse Techniken in chromaffinen Zellen 1. Darüber hinaus, indem sie eine Möglichkeit, sich für einzelne Vesikel-Auflösung der Proteinmaschinerie in Exozytose beteiligt zu ermöglichen, bleibt die chromaffinen Zellen ein leistungsfähiges Modell, um die Fragen der Vesikel-Fusion-3-Adresse. Cell-Attached-Kapazitätsmessungen wurden zunächst bei der Lösung von Einzel Vesikelfusion während Exozytose 3 genutzt. Exocytose von Vesikeln so klein wie ca. 60 nm im Durchmesser, wurde gezeigt, dass durch die Zellmembran Admittanz Messungen mit der Patch-Clamp-Technik in der Zelle verbundene Konfiguration 4-7 detektiert werden. Zulassung als Maß dafür, wie leicht eine Schaltung oder Vorrichtung ermöglicht, dass ein Strom fließen definiert; es ist das Inverse der Impedanz. Somit Admittanz Messungen liefern ein Verständnis der Membrankapazität. Dies wird durch den Einbau der Vesikelmembran in die Plasmamembran durchgeführt; Dieser Einbau zeigt Veränderungen der OberflächenBereich 8. Jedes Schmelzen Vesikel bewirkt eine schrittweise Erhöhung der Membrankapazität 9,10. Zusätzlich liefert diese Admittanzmessung die Membranleitfähigkeit und der Fusionsporen Leitfähigkeit während einer Exozytose-Ereignis 3. Wie diese Technik ein einzigartiges Werkzeug zur Identifizierung Single-Vesikel Kinetik während Exozytose vorgesehen ist, hat unser Labor vor kurzem angewendet dieses Konzept auf die Endozytose von einzelnen Vesikeln 11,12 zu erkennen. Unser Interesse gilt Clathrin-vermittelte Endozytose (CME), die als Grundkomponente Housekeeping in vielen Zellen 13 und ein Hauptpfad für die synaptische Vesikel Endozytose in neuronalen Anschlüsse 14,15 in Betracht gezogen wurde. CME ist bekannt, biologisch wichtig zu sein, bleiben jedoch seine Kinetik nicht gut verstanden aufgrund technischer Einschränkungen bei der Überwachung Singular endozytischen Veranstaltungen. Angesichts der Ähnlichkeiten in Exozytose Mechanismen zwischen chromaffinen Zellen und Neuronen 1 ist es plausible, dass die Spaltung in chromaffinen Zellen Mechanismen kann wahrscheinlich synaptischen Vesikel Endozytose gelten in Neuronen. Die Cell-Attached-Kapazitätsmessungen verwendet worden, um einzelne endozytischen Ereignisse zu überwachen und zu analysieren, die Spalt Kinetik, die die meisten Methoden nicht lösen können. In unserem Zell angebracht Aufnahmen wird eine Sinuswelle bei 20 kHz über die Haltepotential überlagert, und der Ausgangsstrom wird in Membranleitfähigkeit in einem Kanal und Membrankapazität in den anderen Kanal aus einem Zweiphasenverriegelungsverstärker 16 getrennt 18. Aus den Veränderungen in der Membranleitfähigkeit und die Kapazität kann man die Kinetik der Spaltung Poren, die wahrscheinlich entspricht der röhrenförmigen Membran, die den Hals Internalisierungsfaktor Bläschen an der Plasmamembran-Vesikel vor der Abschnürung verbindet berechnen. Kollektiv, gibt uns diese Technik die Möglichkeit, die regulatorischen Mechanismen der Vesikel Spaltung während CME zu prüfen.

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
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			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
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		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
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			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
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			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="22">
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		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

Die Zelllebensfähigkeit und die Qualität der Dichtung Gigaohm sind entscheidend für die Qualität der Cell-Attached-Kapazität Aufnahmen. Daher ist es wichtig, eine effektive und effiziente Zellkultur vor der elektrophysiologischen Ableitungen und typische lebensfähigen Zellen beschaffen sind in Abbildung 1 dargestellt. Üben und Zeit hilfreich, um eine Giga-Ohm Dichtung mit hoher Qualität sein. Wenn man kann deutlich sehen, Zellverformung, wenn der Patch-Pipette nähert sich der Zelle, wie in Schr...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Methoden für die Cell-Attached-Kapazitätsmessungen in Maus-chromaffinen Nebennierenzell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K Aufrufe.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Serie: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultraschall-gesteuerte Mikroinjektion in der Maus Vorderhirnneuronen In Utero In E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Forschung

Neurowissenschaften

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Maus Neuroanatomische Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-Clamp Kapazitätsmessungen und Ca 2 + Imaging bei Single Nervenendigungen in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Methoden zur intravenösen Selbstverwaltung in einem Maus-Modell

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Methoden zur Analyse und Modulation von NF-kappaB-abhängigen adulte Neurogenese

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

In-vivo-Elektrophysiologische Messungen an Maus Ischiasnerven

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Ein Verhaltensbildschirm für hitzeinduzierte Anfälle in Mausmodellen der Epilepsie

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Zwei Peeling-Methoden zur Isolierung von Photorezeptor-Zellkompartimenten in der Netzhaut der Maus zur Proteinanalyse

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...