D. Les Sames merci Harold G. &amp; Y. Leila Mathers Charitable Foundation et l&#39;Université de Columbia initiatives en sciences et génie. D. Sames et D. Sulzer remercier la Fondation McKnight pour les innovations technologiques dans McKnight Prix neurosciences D. Sulzer grâce NIDA, NIMH, et la Picower et Fondations maladie de Parkinson. H. Zhang grâce NARSAD. RH Edwards remercie la Fondation Michael J. Fox, la National Parkinson Foundation, le NIDA et NiMH. Nous remercions Robert Burke pour la 6-OHDA injections et des conseils, Sonders Mark pour des discussions utiles, Merek Siu pour la programmation de l&#39;analyse d&#39;imagerie, et Jan Schmoranzer pour le soutien technique avec l&#39;installation de microscopie TIRF.

Cette méthode a été utilisée dans la recherche présentée dans <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">gubernator et al., Science 324 (5933). 1441-1444 (2009)</a> . 1. Préparation des tranches striatales aiguë <ol><li> Avant de préparer aiguë tranches striatales, il faut oxygéner le liquide céphalo-rachidien artificiel (FCSA) et le refroidir avec de la glace pendant au moins 15 minutes avant l&#39;extraction du cerveau. Gardez le froid de la glace ACSF et oxygéné pendant la dissection. ACSF (en 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 à 7.4, 290-295 mOsm. </li><li> Décapitez souris mâle sans anesthésie. </li><li> Extrait de l&#39;ensemble du cerveau de la souris, et le monter sur le plateau vibratome mis sur le vibratome utilisant Krazy Glue ®. L&#39;extraction du cerveau et de montage doit être fait dans les 2 minutes. Pendant ce temps, veillez à conserver le froid de la glace ACSF et oxygénée pour garder le tissu sain. </li><li> Couper des tranches de cerveau coronale striatum à une épaisseur de 250μm entre bregma 1.54 à 0.62 1. Trois tranches entières seront obtenus qui peuvent ensuite être coupée en 6 tranches moitié avec une aiguille. </li><li> Incuber les coupes de cerveau de l&#39;ACSF oxygénée à température ambiante pendant au moins 1 heure avant le chargement de la sonde. Tranches d&#39;affichage bonne FFN511 chargement et rester actifs pour l&#39;imagerie jusqu&#39;à 4 heures après la préparation tranche. </li></ol> 2. Chargement FFN511 <ol><li> Préparer FFN511 solution de chargement (10 uM FFN511 dans ACSF); également préparer 100 microns ADVASEP-7 dans l&#39;ACSF. Toutes les solutions doivent être fraîchement préparés et oxygéné au moins 15 minutes avant le chargement dans les tranches. </li><li> Individuellement incuber tranche avec FFN511 solution de chargement pendant 30 minutes à température ambiante. </li><li> Retirez ensuite le colorant liée au tissu extracellulaire en incubant la tranche chargé dans oxygénée 100 microns ADVASEP-7 ACSF pendant 30 minutes à température ambiante 2. Les tranches sont maintenant prêts pour l&#39;imagerie. </li></ol> 3. Imagerie FFN511 dans les tranches de cerveau L&#39;imagerie de FFN511 dans les tranches est réalisée en utilisant un microscope laser multiphotonique à balayage. Nous utilisons un Prairies Ultima multiphotonique microscope à balayage laser (Nous étions en utilisant un Zeiss LSM 510 NLO multiphotonique microscope à balayage laser, préalablement et certains résultats ont été obtenus avec le microscope Zeiss). <ol><li> Placez la tranche FFN511-chargés dans la chambre d&#39;enregistrement (RC-27L, Warner) et superfuse avec ACSF oxygénée à un débit de 1-2 ml par minute. Ensuite, placez une harpe de platine avec des cordes en nylon sur le dessus de la tranche afin de minimiser le mouvement pendant l&#39;expérience. Pour réduire davantage le mouvement, permettre à la tranche de se stabiliser pendant au moins 10 minutes dans la chambre avant de l&#39;imagerie. </li><li> Visualisez et repérez le striatum dorsal avec la luminescence champ lumineux sous un objectif à immersion d&#39;eau 10x. </li><li> Place et la position des électrodes bipolaires de tungstène tordus dans cette région, si l&#39;exécution d&#39;une expérience de stimulation. La zone d&#39;imagerie idéale se situe à moins de 300 um entre les deux extrémités de l&#39;électrode bipolaire de stimulation. Placez cette région au centre du champ visuel. </li><li> Image FFN511 marqué terminaux striatum sous un 63x (0,9 NA) L&#39;objectif ultraviolets immersion dans l&#39;eau. FFN511 est excité à 760 nm avec un laser Mai Tai de la physique et la fluorescence spectrale optimale des terminaux striatum est vu à travers un filtre passe-bande (480-520 nm). Les images sont capturées en 12 bits avec le format 75 x 75 um régions d&#39;intérêt à la résolution 512 x 512 pixels. Pour stimuler les expériences de décoloration, pour compenser l&#39;axe z décalage, un z-série d&#39;images 5-7, séparés par une um dans le plan z, est obtenue pour chaque période de temps. </li></ol> 4. FFN511 décoloration L&#39;étiquette peut être FFN511 décolorés, soit par une forte concentration de KCl (nous utilisons 70 mM de KCl), (+)-amphétamine sulfate (AMPH, 20 uM), ou la stimulation électrique. Dans l&#39;expérience de décoloration de KCl, quand une solution de potassium haute ACSF est appliqué à la tranche de cerveau, le FFN511 destains l&#39;étiquette dans les 2 minutes. Enregistrement xyz-t images pour suivre FFN511 décoloration au fil du temps. La section suivante décrit la stimulation électrique dépendant de la dopamine décoloration terminale: <ol><li> Pour suivre FFN511 décoloration au fil du temps, xyz-t images sont enregistrées. Afin de garantir un minimum de mouvement de la tranche (moins de 4 um de l&#39;axe z) et aucun photoblanchiment par les images laser de contrôle, des séries chronologiques de la Z-stack doit être obtenu au moins 5 minutes avant la stimulation. Sinon, ajuster la puissance du laser. </li><li> Suite à ces images de contrôle, de continuer l&#39;imagerie xyz-t tandis commencer la stimulation à des fréquences de 1, 4 ou 20 Hz. Les stimuli à 1, 4 ou 20 Hz (300 ms x 1 mA) sont appliquées dans le striatum localement par un isolateur de relance Iso-Flex déclenchée par un maître-8 générateur d&#39;impulsions (AMPI, Jérusalem, Israël) en utilisant des électrodes bipolaires. Afin de minimiserla variation de la dépolarisation des sites de libération, nous avons utilisé un protocole de stimulation appliquant 150% de l&#39;intensité de stimulation maximale déterminée par voltampérométrie cyclique. </li><li> Utilisez un logiciel approprié pour l&#39;analyse d&#39;imagerie. Dans notre laboratoire, nous utilisons l&#39;image J (Wayne Rosband, National Institutes of Health, Rockville, MD) et personnalisée écrite logiciels en IDL (Research Systems, Boulder, CO) afin de quantifier la fluorescence lacrymaux et les changements avec temps3. </li></ol> Résultats Represenative La figure 1 montre le chargement dans FFN511 tranche striatum est maintenant terminée. Images représentant l&#39;aide de l&#39;objectif 10x et 60x sont présentés dans la Fig.1. Le marquage sélectif des terminaux de dopamine peut être décolorés par 20 uM amphétamines. La figure 2 montre décoloration des FFN511 étiquetage par le KCl élevé. La figure 3 montre décoloration des FFN511 étiquetage par stimulation électrique locale. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Figure 1" /> Figure 1 FFN511 étiquettes terminaux de dopamine dans vivons cortico-striatale tranches aiguë (A) dans l&#39;étiquetage par FFN511 aiguë direct cortico-striatale tranche:.. Étiquetage abondant dans le striatum (STR), l&#39;étiquetage clairsemés dans le cortex (CTX), et aucune étiquette corps calleux (CC). Barre d&#39;échelle: 100. Um (B) Décoloration des FFN511 du striatum par l&#39;amphétamine. Panneaux gauche: avant d&#39;amphétamines; panneaux à droite: après 20 minutes de 20 uM amphétamines. Barre d&#39;échelle: 10 um. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Figure 2" /> Figure 2. Décoloration des FFN511 étiquetage par le KCl élevé. Étiquetage FFN511 est décolorée dans les 2 min d&#39;application de 70 mM de KCl dans l&#39;ACSF. Barre d&#39;échelle: 10 microns. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Figure 3" /> Figure 3. Fonction de la fréquence de décoloration des FFN511 étiquetage dans le striatum. (A) la stimulation locale à 4 Hz entraîné décoloration des bornes. La stimulation a commencé à t = 0. Barre d&#39;échelle: 5 um (B) Décoloration des FFN511 à 4 Hz est Ca 2 + - et dépendant de la fréquence.. Contrôles reçu aucune stimulation (153 points lacrymaux de 3 tranches). Décoloration avec du chlorure de cadmium (200 uM) était identique à des contrôles non stimulées (475 points lacrymaux de 5 tranches). Les courbes de décoloration pour chaque fréquence de stimulation ont été ajustées par une fonction de décroissance exponentielle simple et demi-vie (t 1 / 2) Les valeurs calculées comme τ x 0,693 (1 Hz: 765 points lacrymaux, de 9 tranches, 4 Hz: 410 points lacrymaux de 7 tranches, 20 Hz: 416 points lacrymaux de 6 tranches). S&#39;il vous plaît se référer à la micrographie vidéo à partir de 2-microscopie biphotonique des FFN511 décoloration lors de l&#39;exocytose dans une préparation de tranches de cerveau du striatum.

<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.

Dans cette vidéo, nous démontrons une méthode pour visualiser la neurotransmission dopaminergique optiquement en utilisant des analogues fluorescents. FFN511 est la première génération de FFNs nous avons développé. Bien qu&#39;il ait été conçue en ciblant les neurones du transporteur vésiculaire monoamine (VMAT2) qui transporte les neurotransmetteurs inhibiteurs de la monoamine du cytoplasme dans des vésicules synaptiques, et les labels spécifiquement terminaux de dopamine dans le striatum et les catécholamines présumée et / ou les terminaux de la sérotonine dans le cortex (comme indiqué dans la science papier), une période de chargement appropriée est essentielle pour la spécificité puisque FFN511 est relativement hydrophobe. Nous avons constaté que d&#39;incubation de plus de 40 minutes se traduira par un marquage non spécifique approfondie en tranches striatales. La spécificité peut être directement déterminée par de décoloration en utilisant une forte concentration de KCl, ce qui devrait entraîner la libération du FFN dans les vésicules synaptiques fonctionnelles. L&#39;exposition de la tranche d&#39;FFN511 pour les moins de 15 minutes entraînera signal fluorescent faible en raison de l&#39;insuffisance de chargement du colorant dans les terminaux. Dans ce protocole, nous utilisons 100 microns ADVASEP-7 pour éliminer le colorant liée au tissu extracellulaire. Cette étape n&#39;est pas nécessaire, mais si elle est omise, le temps de purge de l&#39;ACSF a besoin d&#39;être prolongée. En résumé, selon la préparation que vous avez choisi, vous aurez besoin de modifier la période de concentration et de chargement pour déterminer l&#39;étiquetage optimal.

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		<th>Catalogue Number</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>
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<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>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
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dopamine release at individual presynaptic terminals visualized with ffns

Le système nerveux transmet des signaux entre les neurones via la libération de neurotransmetteurs lors de la fusion des vésicules synaptiques. Pour observer l&#39;absorption et la libération de neurotransmetteurs différents terminaux présynaptiques directement, nous avons conçu fluorescentes faux neurotransmetteurs comme substrats pour le transporteur de vésicules synaptiques inhibiteurs de la monoamine. L&#39;utilisation de ces sondes pour la libération de dopamine dans le striatum l&#39;image, nous avons fait plusieurs observations pertinentes à la plasticité synaptique. Nous avons constaté que la fraction de vésicules synaptiques libérant des neurotransmetteurs par stimulus était dépendante de la fréquence de stimulation. Une cinétique distincte «réserve» de la population de vésicules synaptiques n&#39;a pas été observé dans ces conditions expérimentales. Une hétérogénéité dépendant de la fréquence des terminaisons présynaptiques a été révélé que dépendait en partie sur les récepteurs dopaminergiques D2, indiquant un mécanisme de dépendant de la fréquence de codage de la sélection présynaptique. Hui Zhang et Niko G. gubernator contribué également à ce travail.

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

Un nouveau moyen de mesurer la neurotransmission dopaminergique optiquement en utilisant des analogues fluorescents.

La libération de dopamine au niveau individuel terminaisons présynaptiques visualisées avec 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

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JoVE Journal

Biology

24.7K vues.  Columbia University. Un nouveau moyen de mesurer la neurotransmission dopaminergique optiquement en utilisant des analogues fluorescents.

Vidéo: 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.

Terminaux de chargement nerveuse chez la drosophile avec des indicateurs de calcium

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 ...

Recherche

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.

Étiquetage CSEh et hMSCs d&#39;oxyde de fer nanoparticules pour les non-invasives dans le suivi in ​​vivo avec IRM

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.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

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.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

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.

Modèle murin de la maladie de Parkinson: de lésion dopamine 6-OH au test comportemental

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.

Électrofusion cellulaire visualisé par microscopie à fluorescence

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 - transcriptome à l&#39;échelle de cartographie des protéines-ARN Interactions avec résolution nucléotides individuels

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.

Préparation individuelle Drosophila Œuf Chambres pour l&#39;imagerie en direct

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.

Interactions protéine-protéine visualisés par fluorescence bimoléculaire complémentation en protoplastes de tabac et des feuilles

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.

L&#39;imagerie de fluorescence avec un nanomètre Précision (FIONA)

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