Lösungen <ul><li> Intrazelluläre Lösung für Ganglienzellen (in mm): 100 K-Gluconat, 8 KCl, 1 MgCl 2, 1 EGTA, 10 HEPES, 4 ATP, .5 GTP, 90 uM Sulforhodamine B (für die Färbung), auf pH = 7,4 mit angepassten KOH </li><li> Intrazelluläre Lösung für bipolare Zellen (in mm): 90 K-Gluconat, 8 KCl, 1 MgCl 2, 10 BAPTA, 10 HEPES, 4 ATP, .5 GTP, 90 uM Sulforhodamine B (für die Färbung), auf pH = 7,4 mit angepassten KOH </li><li> Extrazelluläre Ringer-Lösung (in mM): 112 NaCl, 5 Glucose, 5 HEPES, 2 KCl, 2 CaCl 2, 1 MgCl 2, eingestellt auf pH = 7,75 mit NaOH. Oxygenierung der Lösung ist nicht notwendig. </li></ul> Bereiten Sie die Aufnahme Kammern Unsere Aufnahme Kammer besteht aus einem Mikroskop-Objektträger mit einem Klebstoff auch in die kleinen Silikon-Blöcke aus einem anderen Klebstoff schneiden gut aufgestellt, um Stabilität für die Gewebeschnitte Verfügung zu stellen. Vorbereitung der Netzhaut Scheiben Um zu vermeiden, Bleichen der Photorezeptoren ist die Zerlegung in IR-oder dim rotes Licht Licht durchgeführt. Wenn man interessiert sich für die Aufnahme Antworten von der Stange Weg, sollte nur IR-Licht verwendet (IR Präpariermikroskop und kein Licht ausgesetzt wird. Zusätzlicher IR-Brillen für Verfahren unter dem Mikroskop nicht getan werden sollte verwendet werden). Wenn man vor allem in der Aufnahme Kegel Licht Reaktionen, dim red room Licht oder eine rote Blitzlicht interessiert ist, kann verwendet werden. Das Mikroskop sollte jedoch noch IR Okulare. Wenn Photorezeptor-Eingang ist irrelevant für das Experiment, können Verfahren, die in normalen Raumbeleuchtung durchgeführt werden mit einem normalen Mikroskop. <ol><li> Bereiten Sie zwei Objektträger mit einem Kleber Silikon gut: Erstellen Sie zwei parallele Bänder von Silikonfett (etwa .5 cm Abstand) auf jeder Folie und platzieren Sie ein rechteckiges Stück Filterpapier auf einer Seite der Folie, wo sowohl Fett-Bands (drücken Sie den Filter Papier mit einem Teil einer Rasierklinge so dass es gut ist, um die Folie im Anhang) </li><li> Sacrifice ein Salamander nach festgelegten Protokollen: Das Tier ist in einem Eisbad für 30 Minuten gelegt, dann enthauptet und doppelklicken pithed. </li><li> Entfernen Auge von Salamander Kopf und legen Sie sie unter dem Binokular auf ein Stück Seidenpapier angefeuchtet mit eiskaltem Ringer-Lösung </li><li> Entfernen Sie das Bindegewebe aus dem Auge </li><li> Verwenden Sie Rasierklinge einen kleinen Schnitt in der Hornhaut machen </li><li> Halten Sie schneiden Hornhaut mit einer Pinzette entfernen, während sie mit einer Schere entlang der Bindung an das Auge </li><li> Nehmen Sie das Objektiv mit einer Pinzette </li><li> Halten Sie die Blende mit einer Pinzette und schneiden entlang der ora serrata, um die Iris zu entfernen </li><li> Schneiden Sie die Augenmuschel in zwei rechteckige Stücke </li><li> Pick-up eines der Stücke und legen Sie sie Sklera nach oben auf das Filterpapier (stellen Sie sicher, abzuflachen das Stück während Sie es sich sehr genau) </li><li> Heben Sie die Lederhaut vor der Netzhaut, die nun sollte dem Filterpapier befestigt werden </li><li> Schnelles Hinzufügen von eiskalter Ringer-Lösung, die auch </li><li> Wiederholen Sie das gleiche für das zweite Stück </li><li> Verwenden einer Rasierklinge in einem speziell angefertigten Allesschneider bis 200-300 pm breite Scheiben schneiden platziert. </li><li> Verwenden Sie eine Pinzette zur Aufnahme der Scheiben an der Seite des Filterpapiers, schalten Sie sie so, dass die Schichten der Netzhaut sichtbar, und sie auf die Vakuum-Fett-Bands </li><li> Wählen Sie ein Slice, das unbeschädigt aussieht und zeigt horizontale Streifen zeigt die Schichten der Netzhaut (die Schichten sind manchmal schwer zu unter Dissektionsmikroskop zu sehen, wie die Vergrößerung ist nicht hoch genug, aber wenn man einige Schichtung erkennen kann, die Scheibe ist in der Regel gut genug. Die endgültige Entscheidung darüber, ob die Scheibe zu verwenden, jedoch wird, wenn es unter dem aufrechten Mikroskop betrachtet wird). Move it, um die Aufnahme Kammer gleiten durch den Bau einer Ringer Brücke zwischen den beiden Folien </li><li> Stellen Sie sicher, das Filterpapier gegen die Gummi-Blöcke in der Aufnahme Kammer geschoben, so dass die Scheibe nicht gekippt (Ganglienzellen und Photorezeptoren sollte im Fokus zugleich sein) </li></ol> Platzierung des Gewebes in der Aufnahme rig <ol><li> Die Aufnahme Folie unter das Mikroskop gelegt und perfundierten mit Ringer-Lösungen durch eine Schwerkraft Perfusion syste </li><li> Die Zubereitung ist sichtbar mit einem aufrechten Mikroskop mit 10x und 40x Wasserimmersionsobjektiv. </li><li> Auch hier verwenden wir nur IR-Licht, um zu verhindern das Ausbleichen der Photorezeptoren. Ein IR-Kamera ist an den seitlichen Anschluss des Mikroskops, die zu einem TV-Bildschirm angeschlossen ist angebracht. </li></ol> Visuelle Stimulation Visuelle Reize werden in Matlab mit dem Psychtoolbox programmiert und sind auf die Netzhaut durch den oberen Anschluss des Mikroskops mit Hilfe eines Mikroskopie Bild Injektor (MBF bioscience) projiziert. A Bits + + Digital Video Processor (Cambridge Research Systems) verwendet, um eine 14bit Leuchtdichte Maßstab zu erhalten und Synchronisationsspgonize Stimulus Präsentation mit Datenerfassung. Die visuelle Reize in diesem Video verwendet werden helle oder dunkle Balken (width = 460μm) von 100% Kontrast, der für zwei Sekunden auf einem stabilen einheitlichen Hintergrund (vorgestellt Luminanz = 8 * 10 4 Photonen / um / s, Größe: 1,84 x 1.38mm). Elektrophysiologie <ul><li> Mit einer Mikropipette puller: Patch-Elektroden sind aus Borosilikatglas (.. 1,5 mm, ID 84mm OD) gezogen. Für Ganglienzellen sind Widerstände zwischen 2 und 4 MOhm. Für bipolare Zellen, sind höhere Widerstände zwischen 4 und 8 MOhm verwendet. </li><li> Voltage Clamp-Aufnahmen sind mit Hilfe einer Multiclamp 700A (Molecular Devices), Verstärker-und Labor-interne Software in LabVIEW programmiert. </li><li> Drogen sind durch einen 8-Kanal-Perfusion System perfundiert. Die Mikroperfusion Elektrode liegt in der Nähe der Scheibe (das Gewebe sollte sichtbar bewegt, wenn Sie die Durchblutung ein-und ausschalten, aber man muss vorsichtig sein, dass der Druck nicht zu stark, so dass die Scheibe könnte sich lösen von dem Filterpapier) gestellt. Bei Versuchen der Mikroperfusion sollte ständig eingeschaltet sein (Perfusion Control-Lösung, wenn keine Medikamente benötigt werden), so dass die Aufzeichnung nicht durch Einschalten der Perfusion und Ausschalten gestört. </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

Vorteile: <ol><li> Alle Zelltypen sind zugänglich </li><li> Leichte Identifikation von Zelltypen, insbesondere wenn ein Fluoreszenzfarbstoff an die Elektrode zugesetzt </li><li> Pharmakologische Wirkstoffe leicht zu erreichen Zielzellen </li><li> Die Patch-Clamp-Technik erlaubt es, die Rolle der verschiedenen Ionenkanäle in der Netzhaut Berechnungen zu untersuchen. Die gleichen Informationen können nicht durch extrazellulären spike-Aufnahmen oder Aufnahmen mit scharfen Elektroden erzielt werden. </li><li> Die...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Multiclamp 700A</td>
<td>Patch Clamp Amplifier</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SZX9</td>
<td>Dissection Micropscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BX50WI</td>
<td>Upright Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td>
</tr>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lucivid</td>
<td>Microscope image injector</td>
<td>Mbf bioscience</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>8-channel perfusion system</td>
<td>Microperfusion</td>
<td>Parker Hannfin corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Digital Video Processor</td>
<td>Cambridge Research Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Oculars</td>
<td>Other</td>
<td>ITT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive silicone wells</td>
<td>Other</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>20mm diameter, 1.0mm deep</td>
</tr>
<tr>
<td>Membrane Filters</td>
<td>Filter</td>
<td>Milipore</td>
<td>HAWPO1300</td>
<td>.45&#181;m HA</td>
</tr>
<tr>
<td>Borosilicate Glass Capillaries</td>
<td>Electrode glass</td>
<td>World Precision Instruments</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Filters</td>
<td>Filter</td>
<td>Whatman</td>
<td>6789-0402</td>
<td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td>
</tr>
<tr>
<td>IR camera</td>
<td>Micropscope mounted camera</td>
<td>Sony</td>
<td>SPT-M324</td>
<td>Extrawave HAD, B7W video camera</td>
</tr>
<tr>
<td>Picrotoxin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1675</td>
<td>&nbsp;</td>
<tr>
<td>Strychnine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0532</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole-4-acetic acid sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I7013</td>
<td>&nbsp;</td>
<tr>
<td>Larval tiger salamanders</td>
<td>Animal</td>
<td>Charles E. Sullivan (Nashville, TN)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

Wir nutzen die whole-cell Patch-Clamp-Technik, um die synaptische Schaltungen, die zugrunde liegende visuelle Informationsverarbeitung in der Netzhaut zu untersuchen. In diesem Video werden wir Sie durch den Prozess der Durchführung Ganzzellableitungen des Lichts hervorgerufen Ströme der einzelnen Zellen in der Netzhaut schneiden Vorbereitung leiten. Wir nutzen die aquatische Tiger Salamander als Tiermodell. Wir von der Beschreibung der Dissektion des Auges beginnen und zeigen, wie Scheiben für elektrophysiologische Aufzeichnungen sind montiert. Sobald die Scheibe in der Aufnahme Kammer gegeben ist, zeigen wir, wie man whole-cell voltage clamp Aufnahmen durchzuführen. Wir projizieren visuelle Reize auf die Photorezeptoren in der Scheibe, um Licht hervorgerufene aktuelle Antworten zu entlocken. Während der Aufnahmen haben wir versorgen die Scheibe mit pharmakologischen Mitteln, wobei eine 8-Kanal-Perfusion-System ermöglicht es uns, schnell zwischen verschiedenen Agenten zu wechseln. Die Netzhaut Scheibe Vorbereitung ist weithin bekannt für Patch Clamp Messungen in der Netzhaut, insbesondere auf amakrinen oder bipolare Zellen, die nicht zugänglich sind in einer ganzen-mount Vorbereitung zu studieren.

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Dieses Video zeigt den Prozess der whole-cell voltage clamp Aufnahmen in der Netzhaut Scheibe der aquatischen Tiger Salamander. Wir zeigen die Herstellung der Scheibe als auch, wie man Patch Clamp Messungen während visueller Stimulation der Netzhaut führen.

Ganzzellableitungen of Light evozierten exzitatorischen synaptischen Ströme in der Netzhaut Scheiben

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Research

JoVE Journal

Biology

12.7K Aufrufe.  Boston University. Dieses Video zeigt den Prozess der whole-cell voltage clamp Aufnahmen in der Netzhaut Scheibe der aquatischen Tiger Salamander. Wir zeigen die Herstellung der Scheibe als auch, wie man Patch Clamp Messungen während visueller Stimulation der Netzhaut führen.

Serie: Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Horizontale Scheiben Vorbereitung der Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Forschung

Biologie

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Whole Cell Recordings von Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Wesentliche Komponenten des Lichtmikroskops

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Recordings of Neural Circuits Aktivierung in Frei Behaving Tiere

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

Whole-Cell Aufnahme von Calcium Release-Activated Calcium (CRAC) Ströme in menschlichen T-Lymphozyten

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Studieren Proteolyse von Cyclin B an der Single Cell Level in Whole Cell Populationen

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Die Slice-Kultur-Verfahren zur Nach Entwicklung der Zahnkeime In Explantation Kultur

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Verfahren zur Beurteilung der Auswirkungen einer Reihe von Wellenlängen und Intensitäten von Rot / Nah-Infrarot-Licht-Therapie auf oxidativen Stress<em&gt; In-vitro-</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...