Die Autoren bedanken sich bei Laura Diaz, Kevin Phillips, Willa A. Hsueh und King C. Li für ihre hilfreichen Kommentare und technische Unterstützung bei der Entwicklung dieser Methode danken.

Ein. Tierische Vorbereitung und Kältebehandlung <ol><li> Suchen und untersuchen Sie ein 4 ° C kalten Raum, der für die Aufnahme von Labormäusen genehmigt wurde. </li><li> Pre-chill Tierkäfige Nacht im Kühlraum. Die Käfige werden ohne Futter und Einstreu, aber mit einer Flasche Wasser montiert. </li><li> Am Morgen des experimentellen Tag, Ort Mäuse einzeln in jedem der vorgekühlte Käfigen bei 30 min Intervallen. Jeder einzeln eingesperrt Maus sollte in der kalten Raum für fast 4 Stunden bleiben, bevor es um die Imaging-Labor transportiert wird. Sicherstellen, dass die Mäuse, aber mit Zugang zu Wasser Fasten. </li><li> Bei 4 Stunden nach der Kältebehandlung Transport eines Tieres in einer Zeit alle 30 min zur Bildgebung Lab. Dies kann durch Füllen eines Behälters mit Styropor Eis und Platzieren einer vorgekühlten Gehäusekäfigs auf der Oberseite der Eis im Inneren der Schachtel erreicht werden kann. Locker den Deckel auf dem Styropor-Box. </li></ol> 2. Setup Micro-PET/CT Imaging-Workflow <p class=&quot;Jove_content&quot;&gt; In diesem Protokoll micro-PET/CT Bildgebung mit der Siemens Inveon Dedicated PET (DPET) System und Inveon Multimodalität (MM) System (CT / SPECT) im angedockten Modus erreicht. Das Tier wird aus dem MM Eingang, zuerst mit dem CT für anatomische Artikeln abgetastet platziert und dann verschoben wird, um die Mitte des DPET für eine statische F18 PET Akquisition. Damit der Host-Computer für die Durchführung dieser sequentiellen Aufgaben automatisch, wird die folgende &quot;Workflow&quot; mit dem Inveon Acquisition Workplace (IAW) Software vor dem eigentlichen Bildgebungssitzung programmiert. <ol><li> CT Acquisition: Bei einer Ganzkörper-CT-Scan, stellen Strom bei 500 uA, Spannung am 80kV, Belichtungszeit bei 200 ms und 240 Schritte für 240 °-Drehung. Für X-ray-Detektor, wählen Auflösung bei &quot;low-System Vergrößerung&quot; mit 78 mm axial Bildfeld und Einzelbett Modus. Wählen Sie &quot;real time Wiederaufbau&quot; mit dem &quot;Common Cone-Beam Reconstruction&quot;-Methode, so dass die Host-PC Gespräche mitein dediziertes Echtzeit Wiederaufbau Computer (Cobra), um die Aufgabe zu initiieren. </li><li> PET Emission Acquisition: Set 600 Sek. (10 min) für &quot;fixed Scan-Zeit&quot; in der &quot;Erwerb durch die Zeit&quot;-Option. Wählen Sie F-18 als &quot;Studie Isotop&quot; und verwenden 350-650 keV als &quot;Energie&quot;. </li><li> PET Emission Histogramm: Set &quot;dynamische frame&quot; als &quot;schwarz&quot; Daten zu verarbeiten als ein Rahmen für die gesamte Dauer zu statischen Scan zu erreichen. Wählen Sie &quot;3D&quot;, wie das Histogramm Art und wählen Sie &quot;keine Streukorrektur&quot;. </li><li> PET Reconstruction: Verwenden Sie 2D-geordnete Teilmenge Erwartungsmaximierung (OSEM2D) als Rekonstruktionsalgorithmus. </li></ol> 3. Die Injektion von FDG <ol><li> Bestellen Sie eine klinische Paket von 18 F-FDG (10 mCi) von einem regionalen Anbieter für seine Ankunft in der Imaging Lab ~ 30 min vor dem geplanten ersten Injektion. Folgen des Instituts Sicherheitsverfahren zu empfangen und zu überblicken das Paket mit radioaktiven Materials (RAM). </li><li> Mit dem Schutz von einem L-Block Tischplatte Schild, Aliquot der FDG und machen Verdünnungen mit sterilem Kochsalzlösung zur Verfügung gestellt. Die verdünnte Aktivitätskonzentration von FDG sollte bei 200-300 μCi/100 ul für jede Injektion verfügbar. Zeichnen Sie die FDG-Lösung in eine 1 ml Spritze mit 26G 1/2 inch Nadel, und messen die Radioaktivität des gesamten Spritze mit einer Dosiskalibrator. </li><li> Spritzen Sie das Tier, das gerade aus dem kalten Raum (siehe Schritt 1,4) mit 100 ul FDG-Lösung über die intraperitoneale (ip) Weg transportiert wird. Notieren Sie sich die Einspritzzeit. Messen Sie den Rückstand Radioaktivität der Spritze wieder mit dem Dosiskalibrator. </li><li> Legen Sie das Tier zurück in den kalten Käfig in einem Styropor-Kühler mit Eis gehalten. Inkubieren das Tier in kaltem (~ 4 ° C) für 1 Stunde für FDG. </li><li> Berechnen des injizierten FDG Aktivität für jede Maus durch die folgende Formel: Injizierten Aktivität (Ci) = Aktivität in der Spritze vor der Injektion- Aktivität in der Spritze nach der Injektion </li></ol> 4. Micro-PET/CT Imaging <ol><li> Die micro-PET/CT Bildgebung beginnt 1 Stunde nach der FDG Injektion oder 5 Stunden nach der Kältebehandlung. Legen Sie das Tier in einer Narkose Kammer mit 3% Isofluran in Sauerstoff. </li><li> Sobald Anästhesie induziert wird, wird das Tier auf eine Mikro-CT Pellet (Tierbettes) mit seinem Kopf ruht in einem Kegel Gesichtsmaske, die kontinuierlich liefert Isofluran (2%) mit einer Fließgeschwindigkeit von 2 L / min bewegt. Eine elektrische Heizkissen (BioVet, m2m Imaging Corp) wird unter das Tier gelegt zur Aufrechterhaltung der Körpertemperatur. </li><li> Schieben Sie das Tier zum Eingang des MM-Scanner, aktivieren Sie die &quot;Laser&quot;-Symbol in der Symbolleiste, und verwenden Sie das Touchpad-Steuerung, um das Bett zu bewegen, so dass die Brust des Tieres ist an der Kreuzung der horizontalen und vertikalen Laserlinien. In der &quot;Laser ausrichten&quot;-Fenster wählen Sie &quot;ersten Scan-Typ&quot; als CT-Scan und &quot;PET Akquisition im Workflow enthalten&quot; als option. </li><li> Öffnen Sie die &quot;Scout View&quot;-Fenster und erwerben ein Scout view Röntgenaufnahme. Verwenden IAW, um die Position des Tieres Bett einzustellen, so dass das Zentrum des Sichtfeldes CT in der Mitte der Maus Körper (Leber) befindet. Wiederholen Sie diesen Schritt, falls erforderlich. </li><li> Starten Sie den &quot;Workflow&quot; in Schritt 2 eingerichtet. Wenn Optionen auftauchen, wählen Sie eine entsprechende 3D-PET-CT Transformationsmatrix Datei in CT-Rekonstruktion verwendet werden, und wählen Sie eine neu geschaffene Normalisierung Datei für PET-Rekonstruktion ohne Schwächungskorrektur. Das IAW startet dann CT-und PET-Scans nacheinander als programmiert. </li><li> Nach der gesamte Workflow abgeschlossen ist, die 20 bis 25 Mai min dauern, kurz bewerten die Qualität der erworbenen CT-und PET-Bilder mit dem ASIPro VM, ein Mikro-PET-Analyse-Software mit IAW mitinstalliert. Archivieren der Bilddaten zu einer Datenspeichereinrichtung oder die Daten über das Netzwerk an einen Pfosten Bildanalyse Arbeitsstation (siehe Schritt 5) für eine weitere Analyse. </li><li> Release das Tier aus den bildgebenden Systemen und legen Sie es auf eine saubere Gehäusekäfig mit normalen Lebensmittel-und Wasserversorgung für die Erholung bei Raumtemperatur. Die Systeme sind nun bereit für die nächste Tier in der Warteschlange. Beachten Sie die Pflege und Behandlung von post-Bildgebung Tiere sollten des Instituts Vorschriften über &quot;Umgang mit Labortieren mit RAM injiziert&quot; folgen. Beachten Sie auch, dass gebrauchte Nadeln / Spritzen, die Saugkissen, Handschuhe und alle Betten und Fäkalien sollte als radioaktiver Abfall betrachtet werden, Umgang und des Instituts relevant RAM Entsorgungsvorschriften. </li></ol> 5. Post-Imaging-Analyse <ol><li> Offene Inveon Research Workplace (IRW) Software (Siemens) und manuell importieren sowohl CT-und PET-Datensätzen. Co-Register CT-und PET-Bilder in Fenster &quot;Registrierung&quot; Einsatz von Werkzeugen mit dem &quot;General-Analyse&quot; Funktion, und unter dem &quot;Review&quot;-Fenster stellen Sie sicher, eine perfekte Ausrichtung zwischen CT-und PET-Bilder in allen 3 Dimensionen. </li><li>Von &quot;Region of Interest (ROI) Quantifizierung&quot;-Fenster, mit den Referenzen von den Ko-registrierten CT-Bilder zur Verfügung gestellt, identifizieren BAT am interskapularen Bereich des Halses, die vorherrschende kälteinduzierbare BAT in erwachsenen Mäusen, und ziehen Volumen von Interesse (VOI) von BAT auf den PET-Datensatz. Wählen Sie &quot;Voxel Intensity&quot; als &quot;Quantifizierung Type&quot; und notieren Sie die mittlere Radioaktivität innerhalb des VOI als Bq / ml. Ein Kalibrierungsfaktor die Counts / sec umwandelt Bq / ml wurde zuvor durch Abtasten eines Phantoms mit bekannten Radioaktivität errichtet. </li><li> FDG-Aufnahme in BAT wird als Prozentsatz der injizierten Dosis pro Gramm Gewebe (% ID / g) mit Zerfallskorrektur quantifiziert. Die injizierte Dosis ist das Ergebnis von Schritt 3.5, jedoch Becquerel (Bq) Einheit (1 uCi = 37.000 Bq) umgewandelt wird; wir an, dass 1 ml bis 1 g Gewebe entspricht. </li></ol>

<ol>
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A., Jacobsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+FDG+uptake+in+brown+adipose+tissue+in+clinical+patients+by+a+single+dose+of+propranolol.">Reduction of FDG uptake in brown adipose tissue in clinical patients by a single dose of propranolol.</a> Eur. J. Nucl. Med. Mol. Imaging. 34, 1018-1022 (2007).</li><li>Zhao, J., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenesis+is+beta3-+but+not+beta1-adrenergically+mediated+in+rat+brown+fat+cells,+even+after+cold+acclimation.">Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.</a> Am. J. Physiol. 275, 2002-2011 (1998).</li><li>Rowland, D. J., Garbow, J. R., Laforest, R., Snyder, A. 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Keine Interessenskonflikte erklärt.

In dieser Studie wurde eine micro-PET/CT-based Bildgebungsverfahren hat zum Erfassen BAT Aktivitäten in erwachsenen Mäusen, die einfach benötigt eine Kältebehandlung und eine Injektion von handelsüblichen 18 F-FDG entwickelt. Das gesamte Verfahren kann in einem Tag nach einer Behandlung und Imaging-Sequenz, die alle 30 Minuten beginnt, bis alle Tiere behandelt werden und abgebildet durchgeführt werden. Unter den experimentellen Bedingungen umrissen, können insgesamt 10 Mäusen (oder 2 Gruppen von 5 Mä...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Micro-PET/CT Imaging System </td><td> Siemens Medical Solutions USA, Inc. </td><td> Inveon Dedicated PET System und Inveon Multimodalität CT / SPECT System (angedockt) </td><td></td></tr><tr><td> Propranolol </td><td> Sigma </td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td> Sigma </td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope Inc. </td><td></td><td></td></tr><tr><td> C57BL/6J männlichen Mäusen </td><td> Jackson Laboratory </td><td> 000664 </td><td> 3-4 Monate alt </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

Braunem Fettgewebe (BAT) von weißem Fettgewebe (WAT) durch seinen diskreten Ort und einem braun-rote Farbe aufgrund reiche Vaskularisierung und hohe Dichte von Mitochondrien. BAT spielt eine wichtige Rolle im Energie-Ausgaben und nicht zitternd Thermogenese bei neugeborenen Säugetieren sowie den Erwachsenen ein. BAT-vermittelten Thermogenese ist stark von der sympathischen Nervensystems reguliert, überwiegend über β-adrenergen Rezeptor 2, 3. Jüngste Studien haben gezeigt, dass BAT Aktivitäten im menschlichen Erwachsenen negativ mit einem Body Mass Index (BMI) und anderen diabetischen Parametern 4-6 korreliert. BAT hat damit als potenzielles Ziel für anti-obesity/anti-diabetes Therapie, die auf Modulation der Energiebilanz 6-8 vorgeschlagen worden. Während mehrere kalte Herausforderung basierenden Positronenemissionstomographie (PET) Verfahren zum Nachweis von menschlichen BAT 9-13 eingerichtet sind, es im Wesentlichen kein standardisiertes Protokoll für die Bildgebung und quantifendungsbe der BAT in kleinen Tiermodelle wie Mäuse. Hier beschreiben wir eine robuste PET / CT-Bildgebung Methode zur funktionellen Beurteilung der BVT in Mäusen. Kurz gesagt, waren erwachsene C57BL/6J-Mäuse kalt unter nüchternen Bedingungen für eine Dauer von 4 Stunden behandelt, bevor sie eine Dosis von 18 F-Fluorodeoxyglucose (FDG) erhalten. Die Mäuse wurden in der Kälte für eine zusätzliche Stunde post-FDG Injektion blieb, und dann gescannt mit einem kleinen Tier-dedizierten micro-PET/CT System. Die erworbenen PET-Bilder wurden mit der CT-Bilder für anatomische Referenzen Kooperationen registriert und analysiert FDG im interskapularen BAT Bereich BAT Aktivität präsentieren. Diese standardisierte kalt-Behandlung und bildgebende Protokoll wurde durch Testen BAT Aktivitäten wurden in pharmakologischen Interventionen validiert beispielsweise die unterdrückte BAT Aktivierung durch Behandlung der β-Adrenozeptor-Antagonist Propranolol 14, 15 oder der erweiterten BAT Aktivierung durch β3-Agonist BRL37344 16. Das Verfahren described hier zum Auffinden von Wirkstoffen / Verbindungen, die BAT-Aktivität modulieren, oder Gene / Wege, die in BAT Entwicklung und Regulation in verschiedenen präklinischen und grundlegende Studien beteiligt zu identifizieren angewendet werden.

Ein Beispiel für micro-PET/CT Bildgebung von Maus BAT ist in Abbildung 1 gezeigt. Während die CT-Bildgebung liefert anatomische Informationen codiert die PET-Bildgebung die Verteilung und Menge von 18 F-FDG durch den ganzen Körper. Diese Bilddaten können separat betrachtet werden (1A und 1B), fusioniert (1C), oder sich mit einem 3D-Merkmal, wie maximale Intensität (MIP, 1D). Mit Hilfe eines 3D-Bildgebung Werkzeug, ein Volumen von Interesse (VOI), hier der interskapularen BAT Region (durc...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Verfahren zur funktionellen Bildgebung von Maus braunen Fettgewebe (BAT) beschrieben, bei dem kalte-stimulierte Aufnahme von 18F-Fluorodeoxyglucose (FDG) in BAT nichtinvasiv beurteilt mit einer standardisierten micro-PET/CT Protokoll ist. Diese Methode ist robust und sensibel auf Unterschiede in der BAT-Aktivitäten in Maus-Modellen erkennen.

Funktionelle Bildgebung Brown Fat in Mäusen mit FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K Aufrufe.  The Methodist Hospital Research Institute, Houston. Verfahren zur funktionellen Bildgebung von Maus braunen Fettgewebe (BAT) beschrieben, bei dem kalte-stimulierte Aufnahme von 18F-Fluorodeoxyglucose (FDG) in BAT nichtinvasiv beurteilt mit einer standardisierten micro-PET/CT Protokoll ist. Diese Methode ist robust und sensibel auf Unterschiede in der BAT-Aktivitäten in Maus-Modellen erkennen.

Serie: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Hell / Dunkel Transition Test für Mäuse

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Forschung

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Labeling Stammzellen mit fluoreszierenden Farbstoffen zur nicht-invasiven Detektion mit Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Funktionelle Bildgebung mit Verstärkung, Eyetracking und Physiologische Überwachung

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevated Plus Maze für Mäuse

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Montage-, Lade-und Alignment einer analytischen Ultrazentrifuge Messkammerdirektanschluss

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques for Imaging Ca 2 + Signaling in menschlichen Spermien

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Eine alternierende Methode, um die traditionelle NASA Hinterlauf Entladen Modell in Mäusen

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Kartierung der Nachwirkungen der Theta Burst Stimulation auf das menschliche Gehör Cortex mit Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Photodiodenbasierte optische Bildgebung zur Aufzeichnung der Netzwerkdynamik mit Einzelneuronauflösung bei nicht-transgenen Wirbellosen

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...