Ce travail a été soutenu par les Instituts de recherche en santé du Canada (IRSC) Subvention à Wayne S. Sossin. Carole A. Farah est le récipiendaire d&#39;une bourse postdoctorale du Fonds de la Recherche en Santé du Québec (FRSQ) et un Conrad Harrington bourse. Les auteurs tiennent à remercier Joanna Bougie, Margaret Hastings et Margaret Labban de l&#39;aide pour le tournage vidéo et Madeline Richmond Piscine pour aider à la présentation graphique.

1. Préparation et entretien des cultures monocouche cellulaire Sf9 <ol><li> Sf9 culture cellulaire et de la maintenance est effectuée sous une hotte de culture de tissus. </li><li> Les cellules Sf9 sont dérivées de cellules de Spodoptera frugiperda l&#39;ovaire (cellules Sf21) et peut être acheté comme cellules congelées dans les médias Grace chez Invitrogen. </li><li> Placer 8 ml de milieu d&#39;insectes de Grace, complétées (1X), à laquelle 30% de sérum bovin fœtal (FBS) a été ajoutée dans un flacon de 75 cm2 de culture cellulaire avec un col incliné. </li><li> Décongelez le tube congelé des cellules Sf9 rapidement dans un bain à 37 ° C eau en déplaçant le tube avant et en arrière (environ 1-2 minutes). </li><li> Transfert des cellules Sf9 aux 75 cm 2 flacon de culture cellulaire et la plaque de pierre doucement par la main pour distribuer les cellules uniformément. </li><li> Incuber 1-2 heures à 27 ° C jusqu&#39;à ce que les cellules ont attaché. </li><li> Retirer ancien milieu et la remplacer par 10 ml de milieu d&#39;insectes freshGrace avec 30% de FBS. </li><li> Incuber à 27 ° C jusqu&#39;à ce que les cellules sont confluentes. </li><li> Pour maintenir les cultures en monocouche, retirez ancien milieu du ballon confluentes de cellules Sf9 et ajouter 5 ml de milieu de Grace insectes avec 10% de FBS. </li><li> Frapper légèrement sur le côté du flacon à plusieurs reprises de se détacher des cellules Sf9. </li><li> En utilisant une pipette de 10 ml et une pipette, une pipette les médias de haut en bas une fois de douceur, la pulvérisation murale flacon lors du pipetage vers le bas. Transférer les cellules à un tube de 15 ml stériles en polystyrène. </li><li> Ajouter 2 mL de suspension cellulaire Sf9 à 8 ml de milieu de Grace insectes avec du FBS à 10% (dilution 1:05 de maintenir la phase de croissance logarithmique) dans de nouvelles de 75 cm 2 flacon de culture cellulaire et la plaque de pierre doucement par la main. </li><li> Incuber à 27 ° C jusqu&#39;à ce que les cellules sont confluentes. </li></ol> 2. Expression des PKC par fluorescence Taggé dans des cellules Sf9 <ol><li> Pour vivre l&#39;expérience d&#39;imagerie, la culture des cellules Sf9 en 35 mm MatTek disheswith à fond de verre de la culture d&#39;une surface de verre de 14 mm et une épaisseur de lamelle de 0,16 à 0,19 mm. </li><li> PKC taggés avec des protéines fluorescentes sont exprimés dans des cellules Sf9 en utilisant Cellfectin recommandation II réactif de transfection suivants du constructeur avec les modifications détaillées ci-dessous. </li><li> Jour 1: Avant de plaquer les cellules, traitent chaque plat avec un milieu de MatTek insectes de Grace avec du FBS à 10% pendant au moins 30 minutes. </li><li> Comptez cellules Sf9 de l&#39;étape 11 (ci-dessus) en utilisant un hématimètre. </li><li> Plate 0,05 x 10 6 cellules sur chaque lamelle de verre de MatTek dans un volume total de 150 pi. Laisser les cellules se fixer pendant 30 minutes à température ambiante. </li><li> Ajouter 2 mL de Grace complété avec 10% de FBS à chaque plat une mL à la fois lentement pour éviter de détacher les cellules. </li><li> Incuber à 27 ° C pendant la nuit. </li><li> De ce point, utilisez les recommandations du fabricant pour la transfection de cellules Sf9 avec l&#39;ADN plasmidique. </li><li> Jour 2: Transfecter cellules Sf9 avec PKC fluorescence marqués en utilisant le réactif de transfection Cellfectin II. Nous avons souvent co-exprimées dans ce système PKC tagués avec améliorée Green Fluorescent Protein (eGFP) et monomères protéine fluorescente rouge (RDRF) (détails pour la construction du plasmide ont été décrits précédemment 2,5). Expression d&#39;une protéine fluorescente à un moment rendements d&#39;environ 70-80% de cellules exprimant 72 heures post-transfection. La co-expression de deux protéines fluorescentes diminue l&#39;efficacité de transfection à environ 30% de co-expression de cellules. Lorsque co-exprimant eGFP-taggés PKC et RDRF marqués à la PKC, utiliser plus d&#39;ADN plasmidique 2x pour la protéine RDRF-marqués pour l&#39;expression protéique optimal. </li><li> Jouez en live-imagerie 48-72 heures post-transfection (pour l&#39;expression protéique optimal, attendez 72 heures). </li></ol> 3. Expression des PKC par fluorescence Tagged dans les neurones sensoriels aplysie <ol><li> Préparation des cultures de cellules neuronales aplysie a été décrit dans un article de la vidéo par Zhao et 4 collègues. </li><li> PKC taggés avec des protéines fluorescentes sont exprimés dans les neurones sensoriels aplysie en utilisant la procédure détaillée ci-dessous la microinjection. </li><li> Préparer une solution stock de 1% Fast Green (FCF) dans l&#39;eau distillée, filtrer à l&#39;aide d&#39;une seringue et une seringue de 28 mm de filtre (0,20 m). Aliquoter et conserver à -20 ° C. </li><li> Microinjection des neurones sensoriels peuvent être effectuées le jour 1 ou 2 jours après avoir isolé les neurones, mais nous constatons que deux jours d&#39;attente permet de mieux l&#39;adhérence cellulaire à la lamelle. </li><li> Le jour 2, après l&#39;isolement des neurones sensoriels, décongeler une aliquote de vert rapide et filtrer à nouveau en utilisant une seringue et un 28 mm filtre seringue (0,20 m). </li><li> Préparer une solution d&#39;ADN plasmidique dans l&#39;eau distillée contenant 0,5% de vert rapide. Les concentrations d&#39;ADN plasmidique aussi élevées que 0,4 ug / uL peut être utilisé mais il est recommandé de ne pas aller au-dessus de ces valeurs. </li><li> Filtre de l&#39;ADN plasmidique / Fast solution verte en utilisant une seringue de 1 mL et de 4 mm filtre seringue (0,20 m). </li><li> Centrifuger l&#39;ADN plasmidique / Fast solution verte à 16110 xg pendant 15 minutes à l&#39;aide d&#39;un microrocentrifuge. </li><li> Préparer électrodes forte pour la microinjection de neurones aide d&#39;un extracteur de microélectrodes (Sutter Flaming / Brown Micropipette Puller, modèle P-97). En utilisant un filament de la boîte, tirez microélectrodes en verre avec 1 mm de diamètre extérieur, 0,75 mm de diamètre intérieur et 100 mm de longueur à l&#39;aide à paroi mince de verre capillaires avec un filament à l&#39;intérieur (World Precision Instruments TW100F-4). Pour plus d&#39;informations sur des électrodes en tirant microinjection s&#39;il vous plaît se référer à l&#39;électrode livre Sutter. </li><li> Remplissez chaque électrode avec 2 pi d&#39;ADN plasmidique / Fast solution verte en utilisant microloader pointe de la pipette (Eppendorf CA32950-050). </li><li> Transfert du verre plat en bas de MatTek culture contenant des cultures de neurones aplysie à la station de microinjection. </li><li> La station de microinjection compose des éléments suivants: une maison étape de grand verre de tenir les boîtes de culture sur une table anti-vibrations (Kinetic Systems poste de travail anti-vibrations), une stéréo-portée avec éclairage halogène externe, un micromanipulateur (Sutter Huxley Micromanipulateur type mur qui permet un positionnement à la fois grossier et ultra-fines dans les trois axes), un porte-microélectrode reliée à une pompe pneumatique (instruments de précision mondiale PV820 pneumatique Pico-pompe). L&#39;entrée de pression de la pompe pneumatique est relié à un réservoir d&#39;azote comprimé. </li><li> Insérer une microélectrode dans le support de microélectrodes. </li><li> Voir sous un stéréomicroscope, insérez la pointe de la micropipette dans le noyau cellulaire. </li><li> Délivrer des impulsions de pression courte de l&#39;azote (10 à 100 ms en durée, 20 lb / po 2) jusqu&#39;à ce que le noyau devient uniformément vert. </li><li> Incuber les cellules pendant 4 heures à température ambiante et de garder à 4 ° C jusqu&#39;à utilisation. Pour la translocation de PKC optimale, en live-imagerie expérience de 20 à 24 heures après l&#39;injection. </li></ol> 4. Visualisation de la translocation de la PKC dans les cellules Sf9 vie et dans les neurones sensoriels aplysie <ol><li> Pour l&#39;imagerie, nous utilisons un microscope inversé Zeiss LSM510 confocale avec une 200 Axiovert. </li><li> Imagerie protocole est le même pour les cellules Sf9 et pour les neurones sensoriels aplysie, sauf pour les éléments suivants: les cellules Sf9 sont imagés avec un objectif x63 huile d&#39;immersion alors neurones sensoriels aplysie sont imagés avec un objectif x40 immersion dans l&#39;huile. Les cellules Sf9 sont imagés dans une chambre à température contrôlée maintenu à 26-27 ° C alors que les neurones sensoriels aplysie sont imagés à la température ambiante maintenue environ 18-20 ° C. </li><li> Nous utilisons un laser Argon 30 mW (excitation à 488nm) avec une sortie laser de 50% à l&#39;image de PKC tagués avec protéine EGFP et un laser HeNe (excitation à 543nm) de la PKC Tagged Image avec des protéines RDRF. L&#39;argon et lignes laser HeNe sont atténués à 4% et de sortie de transmission de 50% respectivement avant l&#39;imagerie et le sténopé est ajusté à un. </li><li> Il est important d&#39;avoir stabilisé la boîte de culture sur la scène d&#39;imagerie et d&#39;éviter tout mouvement ou vibration. </li><li> Retirer 1 ml de milieux de culture de l&#39;antenne doucement à l&#39;aide d&#39;une pipette 10 ml en laissant 1 ml de volume total dans le plat. </li><li> Prenez des photos dans la section du milieu des cellules où le noyau peut être vu. </li><li> Mettre en place une série de temps de 10-20 images confocales de prendre une photo toutes les 30 secondes. </li><li> Démarrer la série chronologique. </li><li> Après deux photos sont prises (après le point de temps de 30 secondes), ajouter 1 mL de la drogue (2X la concentration) doucement goutte à goutte au-dessus des cellules à l&#39;aide d&#39;une pipette P1000. Le médicament est ajouté en continu pendant environ 30 secondes. </li><li> Certains médicaments induisent une translocation rapide qui peut être visible immédiatement un traitement médicamenteux (à la fois le point 60 secondes) tandis que d&#39;autres induisent une translocation lente qui est seulement 5-10 minutes optimale post-traitement (Film 1 et Film 2). </li><li> Si le médicament doit être emportés, utiliser 2 x 10 ml pipettes pour faire la lessive, le pipetage du tampon de lavage doucement avec une pipette sur un côté du plat et de pipetage jusqu&#39;à les médias de l&#39;autre côté. </li><li> Si le lavage a été efficace et si l&#39;effet de la drogue est réversible, la PKC retourne vers le cytosol dans les 30-60 secondes. </li><li> Enregistrer le film en tant que fichier RMLL. </li><li> Utilisez NIH Image J logiciel pour ouvrir les fichiers LSM et de quantifier les images pré et post traitement médicamenteux. La quantification a été décrit ailleurs 1. </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

Nous avons décrit une technique pour la translocation des PKC l&#39;image par fluorescence marqués en temps réel dans des cellules Sf9 et dans les neurones sensoriels aplysie. Les cellules Sf9 de fournir un système simple à l&#39;image de translocation de PKC, depuis la culture et de les transfecter est assez simple. En revanche, la micro-injection de neurones sensoriels aplysie prend un certain temps à maîtriser, jusqu&#39;à quelques mois. Difficultés liées à cette technique comprennent col...

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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>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
		</table>
		</div>

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

La protéine kinase C (PKC) sont des sérine thréonine kinases qui jouent un rôle central dans la régulation d&#39;une grande variété de processus cellulaires tels que la croissance cellulaire et de l&#39;apprentissage et la mémoire. Il ya quatre familles connues des isoformes de la PKC chez les vertébrés: PKC classiques (α, ßi, βII et γ), le type de roman que je PKC (ε et η), le roman de type II PKC (δ et θ), et des PKC atypiques (ζ et ι ). La PKC classiques sont activés par le Ca 2 + et diacylclycerol (DAG), tandis que le roman de PKC sont activées par le DAG, mais sont Ca 2 +-indépendante. La PKC atypiques sont activés par Ca 2 + ni, ni DAG. En Aplysia californica, notre système modèle pour étudier la formation de la mémoire, il ya trois système nerveux spécifiques isoformes de PKC un de chaque grande catégorie, à savoir le traditionnel PKC Apl I, le type de roman que je PKC Apl II et l&#39;atypique PKC Apl III. PKC sont des lipides kinases activées et donc l&#39;activation des PKC classiques et novatrices en réponse aux signaux extracellulaires a été fréquemment corrélée à la translocation de la PKC cytoplasme vers la membrane plasmique. Ainsi, en visualisant la translocation de la PKC dans le temps réel dans des cellules vivantes est devenu un outil précieux pour élucider les voies de transduction du signal qui conduisent à l&#39;activation de PKC. Par exemple, cette technique a permis d&#39;établir que différentes isoformes de la PKC translocation dans des conditions différentes de médiation types distincts de la plasticité synaptique et que la sérotonine (5HT) activation de la PKC Apl II nécessite la production de deux DAG et l&#39;acide phosphatidique (PA) pour translocation 1-2. Fait important, la capacité de visualiser le même neurone a à plusieurs reprises nous a permis, par exemple, pour mesurer la désensibilisation de la réponse de la PKC dans le détail exquis 3. Dans cette vidéo, nous démontrons chaque étape de la préparation des cultures de cellules Sf9, des cultures de neurones sensoriels aplysie ont été décrites dans un autre article 4 vidéo, en exprimant PKC fluorescence marqués dans les cellules Sf9 et dans les neurones sensoriels aplysie et en direct de la translocation de la PKC imagerie en réponse à activateurs différents en utilisant le laser-microscopie à balayage.

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Dans cette vidéo, nous démontrons la visualisation de la translocation de la PKC dans les cellules vivantes en utilisant PKC fluorescence marqués.

Live-imagerie de la translocation de la PKC dans les cellules Sf9 et dans les neurones sensoriels aplysie

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

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Neuroscience

11.9K vues.  McGill University. Dans cette vidéo, nous démontrons la visualisation de la translocation de la PKC dans les cellules vivantes en utilisant PKC fluorescence marqués.

Vidéo: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neurosciences

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation et mitochondriale de transport: Live in Imaging neurones de l&#39;hippocampe sur des durées longues

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus médiée par manipulation génétique et de visualisation des neurones sensoriels olfactifs In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolyse des composés en cage dans les cils olfactifs des neurones sensoriels

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Identifier les motoneurones extracellulaire d&#39;un Motor Pool musculaire dans<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolation des neurones sensoriels de<em&gt; Aplysia californica</em&gt; Patch pour les enregistrements de serrage des courants glutamatergiques

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tout le mont immunomarquage des récepteurs olfactifs neurones dans le<em&gt; Drosophila</em&gt; Antenne

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Une méthode pour cibler et Isoler Airway-innervant neurones sensoriels chez la souris

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...