D. Sames dank der G. Harold &amp; Leila Y. Mathers Charitable Foundation und der Columbia University Initiativen in Science and Engineering. D. Sames und D. Sulzer danken der McKnight Foundation for The McKnight Technologische Innovationen in Neuroscience-Preis D. Sulzer dank NIDA, NIMH, und die Picower und Morbus Parkinson Foundations. H. Zhang dank NARSAD. RH Edwards dank der Michael J. Fox Foundation, der National Parkinson Foundation, NIDA und NIMH. Wir danken Robert Burke für 6-OHDA-Injektionen und Beratung, Mark Sonders für nützliche Diskussion, Merek Siu für Bildanalyse-Programmierung, und Jan Schmoranzer für technische Unterstützung bei der TIRF-Mikroskopie-Setup.

Diese Methode wurde in der Forschung in gemeldet verwendet <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator et al., Science 324 (5933). 1441-1444 (2009)</a> . 1. Vorbereitung akuten striatalen Scheiben <ol><li> Vor der Zubereitung akute striatalen Scheiben, muss man mit Sauerstoff die künstliche Liquor (ASCF) und Chill es mit Eis für mindestens 15 Minuten vor dem Gehirn Extraktion. Halten Sie das ACSF eiskalt und Sauerstoff während der Präparation. ACSF (in mM): NaCl 125, KCl 2,5, NaHCO 3 26, CaCl 2 2,4, MgSO4 1,3, KH 2 PO 4 0,3, Glucose 10, HEPES 5, pH 7,3-7,4, 290-295 mOsm. </li><li> Enthaupten männliche Maus ohne Narkose. </li><li> Entpacken Sie das ganze Gehirn der Maus, und montieren Sie ihn auf der Vibratom Tablett auf dem Vibratom mit Krazy ® Kleber platziert. Brain-Extraktion und Montage müssen innerhalb von 2 Minuten durchgeführt werden. Während dieser Zeit müssen Sie die ACSF Eis kalt zu halten und mit Sauerstoff zu halten das Gewebe gesund. </li><li> Cut koronalen striatalen Hirnschnitten bei 250μm Dicke zwischen Bregma 1,54 bis 0,62 1. Drei ganze Scheiben wird erhalten, die dann in 6 halbe Scheiben mit einer Nadel geschnitten werden. </li><li> Inkubieren Sie die Hirnschnitten in Sauerstoff ACSF bei Raumtemperatur für mindestens 1 Stunde vor Sonde loading. Slices zeigen gute FFN511 Laden und bleiben für die Bildgebung bis zu 4 Stunden nach der Scheibe Vorbereitung aktiv. </li></ol> 2. Loading FFN511 <ol><li> Bereiten FFN511 loading-Lösung (10 um FFN511 in ACSF); bereiten auch 100 &amp;mgr; ADVASEP-7 in ACSF. Alle Lösungen sollten frisch zubereitet werden und mit Sauerstoff versorgt, mindestens 15 Minuten vor dem Laden in die Scheiben. </li><li> Individuell inkubieren Scheibe mit FFN511 loading Lösung für 30 Minuten bei Raumtemperatur. </li><li> Dann entfernen Sie den Farbstoff gebunden an extrazelluläre Gewebe durch Inkubation der beladenen Scheibe in Sauerstoff 100 &amp;mgr; ADVASEP-7 ACSF für 30 Minuten bei Raumtemperatur 2. Slices sind nun bereit für die Bildgebung. </li></ol> 3. Imaging FFN511 in den Hirnschnitten Imaging von FFN511 in den Scheiben wird mit Multiphotonen-Laser-Scanning-Mikroskop. Wir sind mit einem Prairie Ultima Multiphotonen Laser Scanning Mikroskop (Wir waren mit einem Zeiss LSM 510 NLO Multiphotonen Laser Scanning Mikroskop zuvor und einige Ergebnisse wurden mit dem Zeiss-Mikroskop erhalten). <ol><li> Legen Sie die FFN511 belasteten Scheibe in der Aufnahme Kammer (RC-27L, Warner) und superfuse mit sauerstoffreichem ACSF bei einer Flussrate von 1-2 ml pro Minute. Dann legen Sie eine Platin Harfe mit Nylon-Saiten auf der Scheibe, um die Bewegung während des Experiments zu minimieren. Zur weiteren Minimierung Bewegung, damit die Scheibe für mindestens 10 Minuten in der Kammer vor der Aufnahme zu stabilisieren. </li><li> Visualisieren und suchen Sie das dorsale Striatum mit Hellfeld Lumineszenz unter einem 10x Wasser Immersionsobjektiv. </li><li> Ort und Lage der bipolaren twisted Wolframelektroden in dieser Region, wenn die Durchführung einer Stimulation zu experimentieren. Die ideale Imaging-Bereich ist in 300 um zwischen den beiden Spitzen der anregenden bipolaren Elektrode befindet. Platzieren Sie diesen Bereich in der Mitte des Gesichtsfeldes. </li><li> Bild FFN511-markierten striatalen Klemmen unter einem 63x (0,9 NA) Eintauchen in Wasser UV-Ziel. FFN511 wird bei 760 nm mit einem Mai Tai Laser von Spectral Physik und optimale Fluoreszenz der striatalen Klemmen gespannt wird durch ein Bandpassfilter (480-520 nm) zu sehen. Die Bilder werden in 12-Bit-Format mit 75 x 75 um regions of interest in 512 x 512 Pixel Auflösung erfasst. Für stimuliert Entfärben Experimente ist es, für z-Achse verschieben, ein z-Serie von 5-7 Bildern, von 1 um in der z-Ebene getrennt, Ausgleich für jeden Zeitraum erhalten. </li></ol> 4. FFN511 Entfärbung Die FFN511 Etikett kann entweder durch eine hohe Konzentration von KCl (wir verwenden 70 mM KCl), (+)-Amphetamin Sulfat (AMPH, 20 uM) oder elektrische Stimulation entfärbt werden. In der KCl Entfärben Experiment, wenn eine hohe Kalium-ACSF Lösung der Hirnschnitt angewendet wird, die FFN511 Label destains innerhalb von 2 Minuten. Rekord xyz-t Bilder FFN511 Entfärbung im Laufe der Zeit zu verfolgen. Im Folgenden werden elektrische Stimulation abhängig Dopamin-Terminal Entfärben: <ol><li> So verfolgen FFN511 Entfärbung im Laufe der Zeit sind xyz-t Bilder aufgezeichnet. Um sicherzustellen, minimale Bewegung der Scheibe (weniger als 4 um in z-Achse) und kein Ausbleichen durch den Laser-, Steuer-Zeitreihen Bilder der z-Stapel sollte für mindestens 5 Minuten vor der Stimulation erreicht werden. Andernfalls ist die Laserleistung. </li><li> Im Anschluss an diese Kontrolle Bilder, weiterhin die xyz-t Bildgebung während Start der Stimulation bei Frequenzen von 1, 4 oder 20 Hz. Stimuli bei 1, 4 oder 20 Hz (300 us x 1 mA) sind das Striatum vor Ort durch eine Iso-Flex Stimulus Isolator durch einen Master-8 Impulsgeber (AMPI, Jerusalem, Israel) unter Verwendung von bipolaren Elektroden ausgelöst angewendet. Zur Minimierung derdie Variation in der Depolarisation der Freigabe Seiten haben wir eine Stimulation verwendete Protokoll Anwendung 150% der maximalen Intensität der Stimulation durch Cyclovoltammetrie bestimmt. </li><li> Verwenden Sie geeignete Software für die Bildanalyse. In unserem Labor verwenden wir Image J (Wayne Rosband, National Institutes of Health, Rockville, MD) und speziell geschriebenen Software IDL (Research Systems, Boulder, CO), um die puncta Fluoreszenz und die Änderungen mit Time3 quantifizieren. </li></ol> Represenative Ergebnisse Abbildung 1 zeigt FFN511 Laden in striatalen Scheibe ist nun abgeschlossen. Repräsentative Bilder mit 10x und 60x objektiv sind in Abb. 1 dargestellt. Die selektive Markierung von Dopamin-Terminals kann durch 20 uM Amphetamin entfärbt werden. Abbildung 2 zeigt Entfärbung FFN511 Kennzeichnung durch hohe KCl. Abbildung 3 zeigt Entfärbung FFN511 Kennzeichnung durch lokale elektrische Stimulation. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Abbildung 1" /> Abbildung 1 FFN511 Etiketten Dopamin-Terminals in Live-kortikalen-striatalen akuten Scheiben (A) Markierung von FFN511 in akuten leben kortikalen-striatalen Scheibe:.. Reichlich Kennzeichnung im Striatum (STR), spärlicher Kennzeichnung in Cortex (CTX) und kein Label in Corpus Callosum (CC). Maßstab:. 100 &amp;mgr; m (B) der Entfärbung FFN511 aus dem Striatum von Amphetamin. Left-Panels: vor Amphetamin, rechts-Panels: nach 20 Minuten von 20 uM Amphetamin. Maßstab: 10 pm. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Entfärbung von FFN511 Kennzeichnung durch hohe KCl. FFN511 Kennzeichnung ist innerhalb von 2 min Anwendung von 70 mM KCl in ACSF entfärbt. Maßstab: 10 um. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Frequenzabhängige Entfärbung FFN511 Kennzeichnung im Striatum. (A) Lokale Stimulation bei 4 Hz ergab Entfärbung von den Terminals entfernt. Stimulation begann bei t = 0 ist. Maßstabsbalken: 5 m (B) Entfärbung von FFN511 bei 4 Hz ist Ca 2 + - und frequenzabhängig.. Kontrollen erhielten keine Stimulation (153 puncta von 3 Scheiben). Entfärbung mit Cadmiumchlorid (200 uM) war identisch mit unstimulierten Kontrollen (475 puncta von 5 Scheiben). Die Entfärbung Kurven für jede Stimulationsfrequenz wurden von einem einzigen exponentiellen Funktion und die Halbwertszeit (t 1 / 2) fit Werte berechnet τ x 0,693 (1 Hz: 765 puncta von 9 Scheiben, 4 Hz: 410 puncta von 7 Scheiben, 20 Hz: 416 puncta von 6 Scheiben). Bitte beachten Sie die Video-Aufnahme von 2-Photonen-Mikroskopie von FFN511 Entfärbung während Exozytose in einem striatalen Hirnschnitten Vorbereitung.

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

In diesem Video zeigen wir eine Methode, um Neurotransmission optisch sichtbar unter Verwendung von fluoreszierenden Dopamin-Analoga. FFN511 ist die erste Generation der FFNs wir entwickelt haben. Es war zwar indem sie auf die neuronale vesikulären Monoamin-Transporter (VMAT2), die Monoamin-Neurotransmittern führt aus dem Zytoplasma in synaptische Vesikel, insbesondere Etiketten Dopamin im Striatum und vermutet, Katecholamin-und / oder Serotonin-Terminals in der Hirnrinde (wie in der Wissenschaft gezeigt ausgelegt Papier), wird eine entsprechende Belastungsdauer entscheidend für die Spezifität, da FFN511 ist relativ hydrophob. Wir fanden, dass die Inkubation länger als 40 Minuten wird in umfangreichen spezifische Färbung in striatalen Scheiben führen. Die Spezifität kann unkompliziert durch Entfärbung mit einer hohen Konzentration von KCl, die die Freisetzung von FFN innerhalb funktionaler synaptischen Vesikeln sollte Ursache ermittelt werden. Die Exposition der Scheibe zu FFN511 für weniger als 15 Minuten wird in schwachen Fluoreszenzsignal aufgrund unzureichender Belastung der Farbstoff in den Terminals führen. In diesem Protokoll, verwenden wir 100 &amp;mgr; ADVASEP-7, um den Farbstoff gebunden an extrazelluläre Gewebe zu entfernen. Dieser Schritt ist nicht notwendig, aber wenn es weggelassen wird, muss die Auswaschzeit in ACSF verlängert werden. Zusammenfassend, abhängig von der Vorbereitung Sie gewählt haben, werden Sie brauchen, um die Konzentration und Belastungsdauer zu modifizieren, um eine optimale Kennzeichnung zu bestimmen.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
<tr>
<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
<tr>
<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
</tr>
<tr>
<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
<td>&nbsp;</td>
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dopamine release at individual presynaptic terminals visualized with ffns

Das Nervensystem überträgt Signale zwischen den Neuronen über die Freisetzung von Neurotransmittern im synaptischen Vesikel Fusion. Zur Beobachtung Neurotransmitter Aufnahme und Abgabe von einzelnen präsynaptischen direkt, haben wir fluoreszierende falsche Neurotransmitter als Substrate für die synaptische Vesikel Monoamin-Transporter. Mit diesen Sonden Bild Freisetzung von Dopamin im Striatum, machten wir einige Beobachtungen relevant für synaptische Plastizität. Wir fanden, dass der Anteil von synaptischen Vesikeln Freisetzung von Neurotransmittern pro Stimulus abhängig von der Reizfrequenz war. Ein kinetisch unterschiedliche &quot;Reserve&quot; synaptischer Vesikel Bevölkerung war nicht unter diesen experimentellen Bedingungen beobachtet. Eine Frequenz-abhängige Heterogenität der präsynaptischen zeigte sich, dass abhängig war zum Teil auf D2 Dopamin-Rezeptoren, was auf einen Mechanismus für die frequenzabhängige Codierung der präsynaptischen Auswahl. Hui Zhang und Niko G. Gubernator trugen gleichermaßen zu dieser Arbeit.

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Ein neues Mittel, um Neurotransmission optisch messen unter Verwendung von fluoreszierenden Dopamin-Analoga.

Freisetzung von Dopamin an den einzelnen präsynaptischen visualisiert FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K Aufrufe.  Columbia University. Ein neues Mittel, um Neurotransmission optisch messen unter Verwendung von fluoreszierenden Dopamin-Analoga.

Serie: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Loading Drosophila Nerve Terminals mit Calcium-Indikatoren

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primäre Dissoziierte Mittelhirn Dopamin Zellkulturen aus Nager Neugeborene

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Mausmodell für Morbus Parkinson: von 6-OH Dopamin Läsion Behavioral-Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Zell Elektroschweißmuffe Visualisierte mit Fluoreszenz-Mikroskopie

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - Transkriptom-weiten Mapping von Protein-RNA Wechselwirkungen mit Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Der Ausarbeitung der einzelnen Drosophila Ei Chambers für Live-Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluoreszenz-Imaging mit One-Nanometer-Genauigkeit (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...