Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (CIHR) Concessione di S. Wayne Sossin. Carole A. Farah è il destinatario di una borsa di studio post-dottorato dal Fonds de la Recherche en Santé du Québec (FRSQ) e una borsa di studio Conrad Harrington. Gli autori desiderano ringraziare Joanna Bougie, Margaret Hastings e Margaret Labban aiuto per le riprese video e Madeline piscina Richmond per aiutare con la panoramica grafica.

1. Preparazione e manutenzione di colture cellulari monostrato SF9 <ol><li> SF9 coltura cellulare e la manutenzione viene eseguita in una cappa di coltura di tessuti. </li><li> SF9 cellule derivano da cellule ovariche di Spodoptera frugiperda (Sf21 cellule) e può essere acquistato come cellule congelate nei mezzi di comunicazione di Grace da Invitrogen. </li><li> Luogo 8 mL di medium insetti Grace, integrati (1X), per cui il 30% di siero fetale bovino (FBS) è stato aggiunto in un centimetro 75 2 fiasco coltura cellulare con un collo inclinato. </li><li> Scongelare il tubo ghiacciato di SF9 cellule rapidamente in un bagno d&#39;acqua a 37 ° C il tubo muovendo avanti e indietro (circa 1-2 minuti). </li><li> Trasferimento SF9 celle a centimetri 75 2 fiasco di coltura cellulare e placca rocciosa delicatamente a mano per distribuire le cellule in modo uniforme. </li><li> Incubare per 1-2 ore a 27 ° C fino a quando le cellule sono collegati. </li><li> Rimuovere media vecchi e sostituirli con 10 ml di medium insetti freshGrace con 30% FBS. </li><li> Incubare a 27 ° C fino a quando le cellule sono confluenti. </li><li> Per mantenere colture monostrato, rimuovere medio vecchio pallone di confluenza di SF9 cellule e aggiungere 5 ml di medium insetti di Grace con il 10% FBS. </li><li> Toccare leggermente sul lato del pallone più volte a staccare SF9 cellule. </li><li> Usando una pipetta 10 ml e una pipetta, pipetta i media una volta su e giù dolcemente, spruzzando muro fiasco durante il pipettaggio in giù. Trasferire le cellule ad una sterile 15 ml tubo polistirolo. </li><li> Aggiungere 2 ml di sospensione cellulare SF9 a 8 ml di terreno insetti di Grace con il 10% FBS (diluizione 1:5 per mantenere la fase di crescita log) nel nuovo 75 cm 2 fiasco di coltura cellulare e placca rocciosa delicatamente a mano. </li><li> Incubare a 27 ° C fino a quando le cellule sono confluenti. </li></ol> 2. Espressione di PKCS fluorescente Tagged in SF9 cellule <ol><li> Per le immagini live-esperimento, della cultura SF9 celle da 35 mm cultura MatTek disheswith fondo di vetro una superficie di vetro di 14 mm e uno spessore di 0,16 coprioggetto a 0.19 mm. </li><li> PKCS taggati con proteine ​​fluorescenti sono espressi in SF9 cellule utilizzando Cellfectin raccomandazione transfezione II reagente a seguito del costruttore con le modifiche riportate di seguito. </li><li> 1 ° giorno: Prima di placcatura le cellule, il trattamento di ogni piatto MatTek con il mezzo di insetti di Grace con il 10% FBS per almeno 30 minuti. </li><li> Conte SF9 cellule dal punto 11 (sopra) utilizzando un emocitometro. </li><li> Piastra 0,05 x 10 6 cellule per ogni vetrino di vetro MatTek in un volume totale di 150 microlitri. Permettono alle cellule di attaccare per 30 minuti a temperatura ambiente. </li><li> Aggiungere 2 ml di Grazia è integrato con il 10% FBS per ogni ml piatto 1 alla volta lentamente per evitare di staccare le cellule. </li><li> Incubare a 27 ° C durante la notte. </li><li> Da questo punto in poi, l&#39;uso raccomandazione del produttore per la transfezione di SF9 cellule con DNA plasmidico. </li><li> 2 ° giorno: SF9 trasfezione delle cellule con PKCS fluorescente marcato mediante Cellfectin reagenti di trasfezione II. Abbiamo spesso co-espressi in questo sistema PKCS taggati con maggiore proteina verde fluorescente (eGFP) e monomerico proteina fluorescente rossa (MRFP) (dettagli per la costruzione plasmide sono state precedentemente descritte 2,5). Espressione di una proteina fluorescente in un momento produce circa il 70-80% che esprimono celle 72 ore dopo la trasfezione. Co-espressione di due proteine ​​fluorescenti diminuisce l&#39;efficienza di trasfezione a circa il 30% di co-cellule che esprimono. Quando co-esprimono eGFP-tagged PKC e MRFP-tagged PKC, utilizzare 2x DNA più plasmide per la MRFP-tag proteine ​​per espressione di proteine ​​ottimale. </li><li> Esibirsi dal vivo-imaging 48-72 ore post-trasfezione (per l&#39;espressione delle proteine ​​ottimali, attendere 72 ore). </li></ol> 3. Espressione di PKCS fluorescente Tagged nei neuroni sensoriali Aplysia <ol><li> Preparazione di colture cellulari neuronali Aplysia è stato descritto in un articolo video di Zhao e 4 colleghi. </li><li> PKCS taggati con proteine ​​fluorescenti sono espressi nei neuroni sensoriali Aplysia tramite microiniezione procedura dettagliata di seguito. </li><li> Preparare una soluzione madre di 1% Verde fast (FCF) in acqua distillata, filtrare utilizzando una siringa e una siringa da 28 mm filtro (0,20 micron). Aliquota e conservare a -20 ° C. </li><li> Microiniezione dei neuroni sensoriali può essere eseguita il giorno 1 o giorno 2 dopo aver isolato i neuroni, ma troviamo che aspettare 2 giorni permette una migliore aderenza delle cellule del coprioggetto. </li><li> Il giorno 2 dopo l&#39;isolamento dei neuroni sensoriali, disgelo un&#39;aliquota di Green veloce e filtrare di nuovo con una siringa e una siringa da 28 mm filtro (0,20 micron). </li><li> Preparare una soluzione di DNA plasmidico in acqua distillata contenente 0,5% Verde veloce. Le concentrazioni di DNA plasmidico più in alto di 0,4 mg / mL può essere usato ma non è consigliabile andare di sopra di questi valori. </li><li> Filtrare il DNA plasmidico / Fast soluzione verde con una siringa da 1 ml e una siringa da 4 mm filtro (0,20 micron). </li><li> Centrifugare il DNA plasmidico / Fast soluzione verde a 16110 xg per 15 minuti utilizzando un microfonorocentrifuge. </li><li> Preparare elettrodi forte per microiniezione di neuroni con un estrattore microelettrodo (Sutter Flaming / Brown Micropipetta Puller, modello P-97). Utilizzando un filamento scatola, tirare microelettrodi di vetro con 1 mm di diametro esterno, 0,75 mm di diametro interno e 100 mm di lunghezza con sottile parete di vetro capillari con un filamento interno (World Strumenti di precisione TW100F-4). Per ulteriori informazioni sulla tirando elettrodi microiniezione fare riferimento al libro di cucina Sutter elettrodo. </li><li> Riempire ogni elettrodo con 2 ml di DNA plasmidico / Fast soluzione verde utilizzando microloader puntale (eppendorf CA32950-050). </li><li> Trasferire il piatto di vetro MatTek fondo di coltura contenente culture Aplysia neuronali alla stazione di microiniezione. </li><li> La stazione di microiniezione è costituito dai seguenti: una fatta in casa grande palcoscenico di vetro per contenere i piatti della cultura su un tavolo di isolamento dalle vibrazioni (Kinetic workstation vibrazioni Sistemi di isolamento), un impianto stereo-scope alogeni con illuminazione esterna, un micromanipolatore (Sutter Huxley Micromanipolatore Tipo di parete che consente il posizionamento sia grossolana e ultra-sottile nei tre assi), titolare di un microelettrodo collegato ad una pompa pneumatica (Strumenti di precisione mondiale PV820 pneumatico Pico-pompa). L&#39;ingresso di pressione della pompa pneumatica è collegato ad un serbatoio di azoto compresso. </li><li> Inserire un microelettrodo nel supporto microelettrodo. </li><li> Guarda sotto uno stereo-microscopio, inserire la punta della micropipetta nel nucleo delle cellule. </li><li> Fornire impulsi di pressione a corto di azoto (da 10 a 100 ms di durata, 20 lb / in 2) fino a quando il nucleo diventa uniformemente verdi. </li><li> Incubare le cellule per 4 ore a temperatura ambiente e conservare a 4 ° C fino all&#39;utilizzo. Per ottimale traslocazione della PKC, esibirsi dal vivo-imaging esperimento 20-24 ore dopo l&#39;iniezione. </li></ol> 4. Visualizzazione di traslocazione della PKC in Living SF9 cellule e nei neuroni sensoriali Aplysia <ol><li> Per l&#39;imaging, usiamo un microscopio invertito Zeiss LSM510 confocale con un Axiovert 200. </li><li> Imaging protocollo è lo stesso per SF9 cellule e dei neuroni sensoriali Aplysia eccezione dei seguenti: SF9 cellule sono ripreso con un obiettivo x63 olio da immersione, mentre i neuroni sensoriali sono Aplysia ripreso con un obiettivo ad immersione olio x40. SF9 cellule vengono esposte a una temperatura controllata camera mantenuta a 26-27 ° C mentre i neuroni di Aplysia sensoriali vengono esposte a temperatura ambiente mantenuto circa 18-20 ° C. </li><li> Usiamo un 30 mW Argon laser (eccitazione a 488 nm) con uscita laser 50% al PKC immagine taggati con proteine ​​eGFP e un laser HeNe (eccitazione a 543nm) per PKC immagine taggati con proteine ​​MRFP. L&#39;argon e HeNe linee laser sono attenuate al 4% e potenza di trasmissione 50%, rispettivamente prima imaging e il foro stenopeico è regolato a uno. </li><li> E &#39;importante avere il piatto cultura stabilizzato sul palco di imaging e di evitare movimenti o vibrazioni. </li><li> Togliere 1 ml di terreni di coltura dal piatto delicatamente con una pipetta 10 ml lasciando 1 mL volume totale nel piatto. </li><li> Per scattare le foto nella sezione centrale della cellula in cui il nucleo può essere visto. </li><li> Impostare una serie temporale di 10-20 immagini confocale di scattare una foto ogni 30 secondi. </li><li> Inizio della serie storica. </li><li> Dopo 2 immagini sono prese (dopo il tempo di 30 punti secondi), aggiungere 1 ml del farmaco (2X concentrazione) delicatamente goccia a goccia sulla parte superiore delle celle usando una pipetta P1000. Il farmaco è aggiunto continuamente per circa 30 secondi. </li><li> Alcuni farmaci inducono una traslocazione veloce che può essere visibile immediatamente dopo il trattamento farmacologico (al momento 60 punti secondi), mentre altri indurre una traslocazione lento che è a pochi minuti ottimale 5-10 post-trattamento (Movie Movie 1 e 2). </li><li> Se il farmaco deve essere lavato via, usare pipette 2 x 10 ml di fare il lavaggio, pipettaggio il tampone di lavaggio giù delicatamente con una pipetta da un lato del piatto e pipeting il supporto sul lato opposto. </li><li> Se il lavaggio è stato efficace e se l&#39;effetto del farmaco è reversibile, PKC torna al citosol entro 30-60 secondi. </li><li> Salvare il filmato come file LSM. </li><li> Usa immagine NIH J software per aprire i file LSM e quantificare le immagini pre e post trattamento farmacologico. La quantificazione è stata descritta altrove 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>

Nessun conflitto di interessi dichiarati.

Abbiamo descritto una tecnica per traslocazione immagine di PKCS fluorescente taggato in tempo reale in SF9 cellule e nei neuroni sensoriali Aplysia. SF9 cellule fornire un sistema semplice per traslocazione immagine PKC dal coltura e trasfezione loro è molto semplice in avanti. Al contrario, la microiniezione di neuroni sensoriali Aplysia vuole del tempo per padroneggiare, fino a pochi mesi. Difficoltà legate a questa tecnica comprendono l&#39;intasamento degli elettrodi. Filtraggio della soluzione ...

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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>
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<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

Proteina chinasi C (PKCS) sono serina treonina chinasi che svolgono un ruolo centrale nella regolazione una vasta gamma di processi cellulari, come la crescita cellulare e di apprendimento e memoria. Ci sono quattro famiglie note delle isoforme PKC nei vertebrati: PKCS classica (α, βI, βII e γ), nuovo tipo I PKCS (ε e η), romanzo di tipo II PKCS (δ e θ), e PKCS atipici (ζ e ι ). Il PKCS classici sono attivati ​​dal Ca 2 + e diacylclycerol (DAG), mentre il PKCS romanzo sono attivati ​​da DAG, ma sono Ca 2 +-indipendenti. Il PKCS atipici sono attivati ​​né Ca 2 + né DAG. In Aplysia californica, il nostro sistema modello per studiare la formazione della memoria, ci sono tre isoforme specifiche del sistema nervoso PKC uno per ogni categoria principale, cioè la tradizionale PKC ho Apl, il tipo di romanzo che PKC Apl II e il III atipico PKC Apl. PKCS sono lipidi-chinasi attivate, e quindi l&#39;attivazione di PKCS classico e romanzo in risposta a segnali extracellulari è stata spesso correlata con traslocazione della PKC dal citoplasma alla membrana plasmatica. Pertanto, visualizzando traslocazione della PKC in tempo reale in cellule vive è diventato uno strumento prezioso per chiarire le vie di trasduzione del segnale che portano all&#39;attivazione della PKC. Per esempio, questa tecnica ha permesso di stabilire che diverse isoforme della PKC traslocare in condizioni diverse di mediare tipi distinti di plasticità sinaptica e che (5HT) l&#39;attivazione della PKC serotonina Apl II richiede la produzione di entrambi i DAG e acido fosfatidico (PA) per traslocazione 1-2. Soprattutto, la capacità di visualizzare il neurone ripetutamente lo stesso ci ha permesso, ad esempio, per misurare desensibilizzazione della risposta PKC nei minimi dettagli 3. In questo video, dimostriamo ogni fase della preparazione SF9 colture cellulari, colture di neuroni sensoriali Aplysia sono stati descritti in un altro articolo video 4, esprimendo PKCS fluorescente taggato in SF9 cellule e nei neuroni sensoriali e Aplysia live-imaging di traslocazione della PKC in risposta a attivatori diversi utilizzando la microscopia a scansione laser.

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

In questo video, dimostriamo la visualizzazione di traslocazione della PKC nelle cellule viventi utilizzando PKCS fluorescente tag.

Live-imaging di traslocazione della PKC in SF9 cellule e nei neuroni sensoriali Aplysia

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 ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K Views.  McGill University. In questo video, dimostriamo la visualizzazione di traslocazione della PKC nelle cellule viventi utilizzando PKCS fluorescente tag.

Video: 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.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

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

Ricerca

Neuroscienze

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.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

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.

Neuromodulazione e mitocondriale di trasporto: Imaging Live in neuroni dell&#39;ippocampo oltre durate a lungo

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

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto 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 ...

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-mediata manipolazione genetica e la visualizzazione dei neuroni sensoriali olfattivi 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 ...

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.

Extracellulare Identificazione neuroni motori per un pool di motore muscolare in<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.

Isolamento dei neuroni sensoriali<em&gt; Aplysia californica</em&gt; Per Bloccare Registrazioni patch di correnti glutamatergico

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.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

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.

Un metodo per identificare e isolare Airway-innervano neuroni sensoriali nei topi

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

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Nel calcio in Vivo Imaging delle cellule ciliate linea laterale in Zebrafish larvale

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Live-imaging di traslocazione della PKC in SF9 cellule e nei neuroni sensoriali Aplysia

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Isolamento dei neuroni sensoriali

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel

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