Name: in vivo imaging of dauer-specific neuronal remodeling in c. elegans
Uploaded: 2014-09-04T15:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1 Crude Dauer feromone Produzione <ol><li> Grow OP50 E. coli da una singola colonia O / N a 37 ° C sotto agitazione in 100 ml di brodo LB a 200 rpm. NOTA: Questo / cultura O N può essere fatta in anticipo e conservato a 4 ° C. </li><li> Grow N2 C. elegans il 15-20 Giugno piatti cm Petri con NGM agar (vedi ricetta nella tabella 1), testa di serie con 50 ml di OP50 E. coli fino a quando il batterio è quasi esaurito 19. </li><li> Fai 5x 250 ml di S supporti (vedi ricetta nella tabella 1) in 1 L beute 19. NOTA: Questa soluzione può essere effettuato in anticipo. </li><li> Coltivare una cultura di OP50 E. coli in 4 l di brodo LB O / N inoculando con 1 ml di OP50 in E. coli e agitazione a 37 ° C per 16 ore. </li><li> Lavare i nematodi da piastre (passo 1.2) con 1 ml di soluzione S basale (vedi ricetta nella tabella 1) e di trasferimento nematodi 4-5 piastre in ciascuna delle 4 1 L Erlenmeyer palloni con i media S preparata al punto 1.3 (a 4 5 piattiper ogni pallone) 19. </li><li> Centrifugare la OP50 dal punto 1.4 a 650 xg per 10 minuti e rimuovere il surnatante. Risospendere ogni pellet batterico in 10 ml di soluzione S basale. Trasferire la stessa quantità di batteri risospese ad ogni pallone di nematodi. </li><li> Collocare i flaconi in un agitatore orbitale a temperatura ambiente (20 - 25 ° C) e 100 - 150 rpm per 4 - 5 giorni fino a quando la soluzione inizia a cancellare, indicando una deplezione di cibo. </li><li> Una volta che il cibo è esaurito, crescerà un&#39;altra cultura O / N di OP50 E. coli in 4x 1 l di brodo LB. Centrifugare i batteri a 650 xg per 10 minuti e aggiungere una pallina da un pallone a ciascuno dei palloni S multimediali con nematodi. Restituire i palloni S media per l&#39;agitatore orbitale, come al punto 1.7, per altri 3-4 giorni fino a quando il cibo si esaurisce di nuovo. </li><li> Dai ~ 1 ml aliquota della soluzione da ciascuna beuta e stimare la concentrazione nematode da sub-campionamento 1 microlitri da ogni 1 ml aliquota e contando il numero di nematodi. NOTA: L&#39;popolazione di nematodi dovrebbe essere 50.000 - 75.000 nematodi / ml. A questa concentrazione la maggior parte dei nematodi sono dauers. </li><li> Eliminare eventuali fiaschi contaminati. NOTA: La contaminazione si manifesta frequentemente come ife fungine visto durante la fase 1.9. </li><li> Nematodi Centrifugare a 4000 xg per 10 min a 4 ° C. Eliminare il pellet nematode e conservare il surnatante. In alternativa, consultare la discussione per l&#39;utilizzo di questi animali per fare un feromone a bassa potenza. </li><li> Filtrare il surnatante con # 1 carta da filtro Whatman. NOTA: Una boccetta di vuoto sarà accelerare questo processo. </li><li> Inoltre Filtrare il surnatante con unità di filtrazione a vuoto sterile 0,45 micron. NOTA: filtrato può essere conservato O / N a 4 ° C o procedere a 1.14. </li><li> Aggiungere il filtrato in un becher da 2 L con un ancoretta. Mettere in una cappa su una piastra riscaldante scalpore. Portate ad ebollizione mescolando ed evaporare a circa 200 ml. Trasferire la soluzione in un becher da 500 ml. Continuare l&#39;ebollizione fino a circa 50 ml sono left. NOTA: Si deve prestare attenzione durante questa fase in quanto è possibile per la soluzione a bollire sopra. Il colore finale 50 ml è un marrone scuro. In qualsiasi momento durante il processo di cottura, il calore può essere spento e il filtrato O / N conservato a temperatura ambiente oa 4 ° C. </li><li> Lasciate raffreddare e centrifugare per 5 minuti a 1000 x g. Versare il surnatante in un becher da 100 ml e scartare il pellet. </li><li> Lessare il surnatante verso il basso per una crosta marrone. Togliere dal fuoco e rompere la crosta con una spatola. Aggiungere sufficiente etanolo 200 prova a coprire la crosta. Coprire il becher con Parafilm e lasciate riposare O / N a RT. Eliminare l&#39;etanolo in un bicchiere pulito e messo a lato. Aggiungere etanolo fresco per coprire la crosta. Ripetere estrazioni O / N 3 volte. Si riuniscono gli estratti. </li><li> Utilizzare una pompa a vuoto per asciugare gli estratti di etanolo. Se una pompa a vuoto non è disponibile, aggiungere l&#39;estratto etanolico di un becher pulito e caldo a 50 ° C sotto agitazione in una cappa aspirante. NOTA: Una volta che l&#39;etanolo è evaporato un residuo appiccicoso di light colore marrone deve rimanere. Se si usa il riscaldamento, la cura dovrebbe essere presa per non bruciare la soluzione di riscaldamento per troppo tempo. </li><li> Disciogliere il residuo in 10 ml di acqua distillata sterile. Filtro sterilizzare questa soluzione con un filtro a siringa da 0,22 micron e conservare a -20 ° C in aliquote di 1 ml. NOTA: 1 ml di acqua viene utilizzata per sciogliere il residuo per ogni 100 ml di coltura nematode liquido originale. Ad esempio, se una delle fiasche di coltura liquido viene scartato a causa della contaminazione, quindi regolare la quantità di acqua utilizzata per sciogliere il residuo. </li></ol> 2 Determinazione del feromone Dose Response <ol><li> Preparare dauer feromone facendo 6x 5 ml di NGM media con Noble agar senza peptone, e integrato con 50 mg / ml di streptomicina solfato e una serie di dauer feromone (vedi ricetta in Tabella 1) 4, 19. Miscelare i componenti in un 13x100 mm provetta di vetro, bollire sopra un becco Bunsen, e poi versare in tre centimetri piastre di Petri. </li><li> Pesare amtubo icrocentrifuge. Aggiungere 1 ml di O / N coltura di E. coli OP50 al tubo e centrifugare a ~ 17.000 xg in una provetta. Rimuovere tutto il surnatante. Pesare nuovamente il tubo e determinare il peso del pellet batterico. Risospendere il pellet con tampone M9 (vedi ricetta in Tabella 1) per una concentrazione finale di 4% (w / v). Aliquotare 10 microlitri della sospensione batterica sul centro di ciascuna piastra feromone 4. Conservare le piastre O / N a RT. </li><li> Scegli 10-15 gravide N2 wild-type ermafroditi un giorno dopo la muta in età adulta sulle piastre feromoni 4. Conservare le piastre a 25 ° C per 3 - 4 ore. </li><li> Scegli fuori tutti i ermafroditi adulti. Registrare il numero di uova deposte per ogni piatto. Sigillare le piastre con parafilm e ritornare a 25 ° C per 3 giorni 17. </li><li> Contare la percentuale di dauers (Figura 2). Nota: Per entrambi i metodi, è importante notare che dauers (e ad un ex minoretenda non dauers) salirà sulle pareti e sul coperchio delle piastre di Petri. E &#39;quindi importante esaminare tutte le superfici del piatto per nematodi. </li><li> Montare i dati ad una curva dose-risposta e stimare la concentrazione CE 90 del feromone (feromone sufficiente ad indurre il 90% degli animali per formare dauers) come mostrato in Figura 3 Nota:. Usiamo tipicamente un modello di regressione logistica per stimare la CE 90 , ma diversi pacchetti software statistici possono avere metodi alternativi che producono stime simili. </li></ol> 3 Immagini dal vivo di Dauer IL2 neuroni <ol><li> Preparare piatti dauer feromoni come nel protocollo 2 utilizzando il valore EC 90 determinata al punto 2.6. </li><li> Indurre dauers utilizzando i metodi del protocollo 2 con un ceppo che trasporta un giornalista transgene integrato IL2 (myIs13 [P klp-6 :: GFP], myIs14 [P klp-6 :: GFP], myIs16 [P <em&gt; KLP-6 :: tdTomato] o qIs56 [P lag-2 :: GFP]) 12, 20, 21. NOTA: Se si utilizza q Is56, espressione di GFP non sarà visibile nei IL2Qs fino a diverse ore nel molt dauer e alcuni degli eventi di rimodellamento iniziali non saranno visibili. Inoltre, mentre il segnale fluorescente dalla P lag-2 :: GFP transgene è in definitiva più luminoso nelle IL2s durante dauer rispetto ai 6 KLP-reporter fluorescenti P, la P lag-2 :: transgene GFP esprime anche debolmente nei muscoli della testa che può mascherare i bei rami IL2. </li><li> Dopo la deposizione delle uova, rimuovere gli ermafroditi adulti, sigillare le piastre con Parafilm e incubare per 34 ore a 25 ° C 17. NOTA: La lunghezza del tempo per la muta dauer varia leggermente da individuo a individuo. La rapida periodo più di dendrite arborizzazione inizia 4-5 ore dopo l&#39;inizio della muta dauer (39-44hr dopo la deposizione delle uova a 25 ° C) 12. </li><li> Il giorno prima di imaging, costituiscono una soluzione di 10% (w / v) agarosio in tampone M9 con dauer feromone alla concentrazione CE 90. Aliquota circa 100 ml di agarosio su un vetrino da microscopio e rapidamente, ma delicatamente, posizionare un&#39;altra diapositiva in alto per creare una superficie piana agarosio. Dopo l&#39;agarosio si è solidificato, avvolgere le diapositive saran wrap e conservare in una camera umida (scatola punta della pipetta con tovaglioli di carta bagnati). </li><li> Dopo il periodo di incubazione 34 ore, esaminare le piastre di feromone per nematodi che hanno smesso faringeo pompaggio. Scivoli separati in modo tale che una sola superficie di un vetrino ha agarosio su di esso. Scegli uno o più nematodi su un pad agarosio del 10% e 1 μ l di 0,1 micron sferette di polistirene 22. NOTA: Con una popolazione ben sincronizzato, la maggior parte dei nematodi sul piatto entrerà nella muta da L2D a dauer. Tuttavia, ci possono essere variazioni tra individuals. E &#39;quindi importante notare funzionalità aggiuntive (stoma occlusione, faringeo rimodellamento) per stimare la quantità di tempo che gli animali sono stati all&#39;interno della muta dauer (vedi risultati rappresentativi per esempio). </li><li> Determinare l&#39;orientamento (sinistra / destra, dorsale / ventrale) del nematode. Cattura immagini di Z-stack di un dendrite IL2Q ad ingrandimento 100X con la possibile intensità di fluorescenza più basso ogni 15 minuti o quando necessario per l&#39;esperimento specifico. Continuare a catturare immagini fino a quando il nematode ha separato dalla cuticola L2D e immobilizzazione è impossibile o se l&#39;animale si riprende da dauer (faringe comincia pompaggio). Quando non l&#39;imaging attivamente, posizionare il vetrino in una camera umida. NOTA: Usiamo un microscopio epifluorescente con un contrasto di interferenza differenziale di fase e motorizzato (DIC) ottica. </li></ol>

<ol>
	<li>West-Eberhard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental Plasticity and Evolution. , (2003).</li><li>Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dauerlarva,+a+post-embryonic+developmental+variant+of+the+nematode+Caenorhabditis+elegans.">The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.</a> Dev. Biol. 46, 326-342 (1975).</li><li>Riddle, D. L., Albert, P. S., Riddle , D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans II. , 739-768 (1998).</li><li>Golden, J. W., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+dauer+larva:+developmental+effects+of+pheromone,+food,+and+temperature.">The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature.</a> Dev. Biol. 102, 368-378 (1984).</li><li>Riddle, D. L., Wood, W. B. <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 Nematode C. elegans. , 393-412 (1988).</li><li>Fielenbach, N., Antebi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+dauer+formation+and+the+molecular+basis+of+plasticity.">C. elegans dauer formation and the molecular basis of plasticity.</a> Genes Dev. 22, 2149-2165 (2008).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-19 (2007).</li><li>Golden, J. W., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pheromone+influences+larval+development+in+the+nematode+Caenorhabditis+elegans.">A pheromone influences larval development in the nematode Caenorhabditis elegans.</a> Science. 218, 578-580 (1982).</li><li>Butcher, R. A., Fujita, M., Schroeder, F. C., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+pheromones+that+control+dauer+development+in+Caenorhabditis+elegans.">Small-molecule pheromones that control dauer development in Caenorhabditis elegans.</a> Nature Chemical Biology. 3, 420-422 (2007).</li><li>Jeong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+structure+and+biological+activity+of+the+Caenorhabditis+elegans+dauer-inducing+pheromone.">Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.</a> Nature. 433, 541-545 (2005).</li><li>Srinivasan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blend+of+small+molecules+regulates+both+mating+and+development+in+Caenorhabditis+elegans.">A blend of small molecules regulates both mating and development in Caenorhabditis elegans.</a> Nature. 454, 1115-1118 (2008).</li><li>Schroeder, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer-Specific+dendrite+arborization+in+C.+regulated+by+KPC-1/Furin.">Dauer-Specific dendrite arborization in C. regulated by KPC-1/Furin.</a> Curr. Biol. 16, 1527-1535 (2013).</li><li>Procko, C., Lu, Y., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+delimit+shape+changes+of+sensory+neuron+receptive+endings+in+C.+elegans.">Glia delimit shape changes of sensory neuron receptive endings in C. elegans.</a> Development. 138, 1371-1381 (2011).</li><li>Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+alterations+in+sensory+neuroanatomy+of+the+Caenorhabditis+elegans+dauer+larva.">Developmental alterations in sensory neuroanatomy of the Caenorhabditis elegans dauer larva.</a> J. Comp. Neurol. 219, 461-481 (1983).</li><li>Vowels, J. J., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+chemosensory+control+of+dauer+formation+in+Caenorhabditis+elegans.">Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans.</a> Genetics. 130, 105-123 (1992).</li><li>Vowels, J. J., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+chemosensory+defects+in+daf-11+and+daf-21+mutants+of+Caenorhabditis+elegans.">Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans.</a> Genetics. 138, 303-316 (1994).</li><li>Ailion, M., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer+formation+induced+by+high+temperatures+in+Caenorhabditis+elegans.">Dauer formation induced by high temperatures in Caenorhabditis elegans.</a> Genetics. 156, 1047-1067 (2000).</li><li>Neal, S. J., Kim, K., Sengupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromone Signaling. , 273-283 (2013).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Ouellet, J., Li, S., Roy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+is+required+for+both+dauer+maintenance+and+recovery+in+C.+elegans.">Notch signalling is required for both dauer maintenance and recovery in C. elegans.</a> Development. 135, 2583-2592 (2008).</li><li>Blelloch, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gon-1+gene+is+required+for+gonadal+morphogenesis+in+Caenorhabditis+elegans.">The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans.</a> Dev. Biol. 216, 382-393 (1999).</li><li>Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, (2012).</li><li>Nelson, F. K., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+study+of+the+Caenorhabditis+elegans+secretory-excretory+system+using+laser+microsurgery.">Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.</a> J. Exp. Zool. 231, 45-56 (1984).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Singh, R., Sulston, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li></ol>

The authors have nothing to disclose

Un esame della neuroplasticità specifici per dauer e altri cambiamenti morfologici Dauer-associata richiede controllata e affidabile di formazione dauers sia attraverso la mutazione genetica (cioè mutanti, DAF-C) o in caso di esposizione di wild-type animali a feromone. Mentre l&#39;uso di una mutazione genetica per indurre dauers è conveniente, può confondere risultati. Pertanto, i dati raccolti con mutazioni daf-c devono essere successivamente confermati con wild-type animali indotti ad entrare n...

Plasticità fenotipica, tra cui plasticità evolutiva e comportamentale, è importante per adattamenti alle condizioni ambientali avverse ed è considerato una forza trainante nell&#39;evoluzione 1. C. elegans è un organismo modello utilizzato frequentemente per gli studi di sviluppo e neurobiologia. In condizioni ideali, C. elegans si sviluppa attraverso quattro stadi larvali prima di entrare nella fase riproduttiva adulto. Tuttavia, in condizioni di scarsa disponibilità di cibo e di alta densità di popolazione, C. elegans può subire un interruttore di sviluppo e di entrare in una fase di lunga durata e resistente alle sollecitazioni dauer 2. Dauers mostrano molte differenze morfologiche e comportamentali da non dauers che probabilmente mediano questa resistenza allo stress. Gli stimoli ambientali e percorsi genetici che regolano l&#39;ingresso dauer sono stati ampiamente descritti 3, 4, 5, 6, 7. Tuttavia, i meccanismi molecolari che controllano le singole modifiche fenotipiche che si verificano durante formazione dauer sono meno ben compreso. L&#39;esame di fenotipi specifici-Dauer richiede la produzione di animali Dauer. Questo può essere realizzato in tre modi: 1) Picking da una cultura affamato di C. elegans, 2) Uso della formazione dauer costitutiva (daf-c) mutanti, 3) induzione della formazione dauer attraverso feromone purificato. E &#39;relativamente semplice per scegliere dauers da una piastra standard NGM con una C. popolazione elegans che ha esaurito il suo batterica di approvvigionamento alimentare. Nelle nostre mani, una piastra standard NGM originariamente seminato con 50 ml di OP50 Escherichia coli, ha permesso di crescere per 3 giorni e successivamente inoculate con 3 - 4 adulti ermafroditi mantenuta a 25 ° C, si esaurirà la fornitura di cibo entro una settimana e produrre diverse migliaia dauers all&#39;interno di un ulteriore 3 giorni (oltre a numerosi non-dauers affamati). Tuttavia, questo metodo non è soddisfacente per i processi che si verificano durante l&#39;esame muta in dAuer. E &#39;difficile determinare quale delle diverse migliaia di animali su un piatto tipico Starved stanno entrando in dauer. Inoltre, l&#39;uso di una piastra di fame non offre alcuna possibilità di sincronizzazione. L&#39;uso di mutanti daf-c consente la sincronizzazione e la generazione affidabile di dauers. Tuttavia, non vi è alcuna garanzia che una particolare daf-c mutazione non influirà altri fenotipi specifici-Dauer da solo o in combinazione con altre mutazioni. La presenza di un feromone dauer stato implicito Cassada e Russell e successivamente dimostrato da Golden e Riddle. Il feromone dauer da allora è stato caratterizzato chimicamente 2, 8, 9, 10. Feromone purificato è costituito da una miscela complessa di ascarosides, che in forme diverse a regolare entrambi i processi comportamentali e di sviluppo 9, 11. L&#39;uso di un estratto di feromone greggio dauer permette di induzione affidabile controllata di dauers sincronizzati in uno sfondo wild-type. </p&gt; Il sistema nervoso e le cellule gliali associate subiscono rimodellamento durante dauer larva formazione 12, 13, 14. Alcuni di questi cambiamenti sono irreversibili, come ad esempio la ristrutturazione delle cellule gliali durante dauer 13, 14. Tuttavia altri eventi di rimodellamento sono reversibili dopo un ritorno favorevole condizioni ambientali. Ad esempio, una serie di quattro neuroni IL2Q subiscono una rapida e reversibile rimodellamento neuronale durante la formazione dauer tra cui: arborizzazione dei dendriti, un passaggio da singolo dendritiche ai neuroni e assoni multidendritic rimodellamento 12. I metodi consentono qui per imaging attendibile in vivo di neuroplasticità indotta da stress in un organismo modello. I metodi presentati per la produzione e la sperimentazione di greggio dauer feromone sono precedentemente descritti e compilati da diverse fonti 4, 8, 15, 16, 17, 18.

<table><tbody><tr><td>N2 &lt;em&gt;C. elegans&lt;/em&gt; Bristol strain</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>PT2660 myIs13[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] III</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>PT2762 myIs14[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] V</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>JK2868 qIs56[P&lt;sub&gt;lag-2&lt;/sub&gt;::gfp] V</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>OP50 E. coli</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Tritech Research</td><td>60 mm T3308, 35 mm T3501</td><td></td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>3 g NaCl, 17 g agar, 2.5 g peptone in 975 ml H&lt;sub&gt;2&lt;/sub&gt;O autoclaved cooled to 55&amp;deg;C followed by 1 ml of 5 mg/ml cholesterol in ethanol, 25 ml of 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 1 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>S Media</td><td>Various</td><td></td><td>S media consists of S basal solution (1.46 g NaCl, 0.25g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.5 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.25 ml of 5 mg/ml cholesterol in ethanol and H&lt;sub&gt;2&lt;/sub&gt;O to 250 ml. Autoclaved.) To the S basal solution add 2.5 ml of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate&lt;sub&gt;, &lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml, autoclaved), 2.5 ml of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.2 g MnCl2&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O, 0.29 g ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.025 g CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Autoclaved and stored in the dark), 0.75 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, and 0.75 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;.</td></tr><tr><td>Dauer pheromone plates for dose-response assay</td><td>Various</td><td></td><td>Mix 0.085 g Noble agar, 0.5 ml of 0.513 M NaCl and 5 &amp;micro;l of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 ml culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H&lt;sub&gt;2&lt;/sub&gt;O to bring the volume to 4.725 ml.&amp;nbsp; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &amp;deg;C. Add 125 &amp;micro;l 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 50 &amp;micro;l 0.1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 50 &amp;micro;l 0.1 M MgSO&lt;sub&gt;4&lt;/sub&gt; and 50 &amp;micro;l of 5 mg/ml streptomycin sulfate to each tube.&amp;nbsp; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td></tr><tr><td>Microsphere beads 0.1 micron</td><td>Polysciences Inc.</td><td>00876-15</td><td></td></tr><tr><td>M9 buffer</td><td>Various</td><td></td><td>Mix 3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 liter. Sterilize by autoclaving</td></tr></tbody></table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

I meccanismi che controllano lo stress-indotta plasticità fenotipica di animali sono spesso complesse e difficili da studiare in vivo. Un classico esempio di plasticità indotta da stress è la fase dauer di C. elegans. Dauers sono uno stadio larvale di sviluppo alternativo formata in condizioni di basse concentrazioni di cibo batterica e alte concentrazioni di un feromone dauer. Dauers visualizzare ampia plasticità evolutiva e comportamentale. Ad esempio, un set di quattro quadranti interno-labiale (IL2Q) neuroni soggetta ad un esteso rimodellamento reversibile durante la formazione dauer. Utilizzando la ben nota voce di percorsi ambientali di regolazione dauer, un metodo precedentemente stabilito per la produzione di greggio dauer feromone da grandi culture nematodi liquido è dimostrata. Con questo metodo, una concentrazione di 50.000 - 75.000 nematodi / ml di coltura liquido è sufficiente a produrre una molto potente greggio dauer feromone. La potenza feromone greggio è determined da un saggio biologico dose-risposta. Infine, sono descritti i metodi utilizzati per in vivo time-lapse imaging dei IL2Qs durante la formazione dauer.

La produzione di greggio risultati dauer feromoni in un liquido giallo (Figura 1) che è sia caldo e freddo stabile. Aliquote di feromone greggio vengono conservati a -20 ° C indefinitamente senza perdita evidente di attività. In seguito la produzione di feromoni, un unico biotest dose-risposta è sufficiente per una stima approssimativa di potenza. Tuttavia, per la maggior parte degli esperimenti è necessario ripetere il saggio biologico 2 - 3 volte e prendere una media di ciascun esperimento. I ris...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

In Vivo Imaging di Dauer-specifica neuronale ritocca in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

11.2K Views.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

Video: In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Semplici dispositivi microfluidici per<em&gt; In vivo</em&gt; Imaging di<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Ricerca

Bioingegneria

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In Vivo Calcium Imaging in C. elegans Muscoli della parete del corpo

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochimica

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

In vivo imaging di calcio neuronale in C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Neuroscienze

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers Agarose per lungo termine Imaging di calcio<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Gli embrioni utilizzando un microscopio epifluorescente e Software Open Source

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biologia

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Un semplice chip microfluidico per la crescita a lungo termine e l'imaging di Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Valutazione della dinamica e dell'orientamento dei microtubuli nei neuroni della caenorhabditis elegans

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Quantificazione dei livelli di alterazione morfologica e degenerazione dei neuroni dopaminergici in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imaging subcellulare della manipolazione neuronale del calcio in vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

In Vivo Imaging di Dauer-specifica neuronale ritocca in C. elegans

Riepilogo

Esplora Altri Video

Capitoli in questo video

Semplici dispositivi microfluidici per

In Vivo Calcium Imaging in C. elegans Muscoli della parete del corpo

In vivo imaging di calcio neuronale in C. elegans

Microchambers Agarose per lungo termine Imaging di calcio

Imaging C. elegans Gli embrioni utilizzando un microscopio epifluorescente e Software Open Source

Un semplice chip microfluidico per la crescita a lungo termine e l'imaging di Caenorhabditis elegans

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In vivo Valutazione della dinamica e dell'orientamento dei microtubuli nei neuroni della caenorhabditis elegans

Quantificazione dei livelli di alterazione morfologica e degenerazione dei neuroni dopaminergici in Caenorhabditis elegans

Imaging subcellulare della manipolazione neuronale del calcio in vivo