Vorremmo ringraziare Scott Hamilton, ed i membri del laboratorio di O&#39;Halloran per la lettura attenta di questo manoscritto. Ringraziamo anche il nostro anonimo recensore per eccellenti suggerimenti e commenti penetranti.

1. Costruzione di GFP Tagged EGL-4 Animali Esprimere <ol><li> Clonare il traslazionale fusione GFP :: EGL-4 sotto il promotore per l&#39;ODR-3 gene (uso 2678 bp direttamente a monte del codone di inizio): (p) ODR-3 :: GFP :: EGL-4. L&#39;ODR-3 unità promotore espressione nelle coppie neurone amphid: AWA, AWB, AWC e debolmente in ASH. </li><li> Iniettare il plasmide (p) ODR-3 :: GFP :: EGL-4 in di tipo selvatico (N2) gli animali a 50 ng / mL con i co-iniezione utilizzando le normali marcatori della linea germinale tecniche di trasformazione 8: (p) ofm-1: : GFP 9 (25 ng / pl), e (p) ODR-1 :: DsRed 10 (25 ng / pl). L&#39;ODR-1 espressione promotore unità nel AWC e AWB amphid coppie di neuroni, e la-1 ofm espressione promotore unità negli coelomocytes. </li><li> Sottoporre gli animali transgenici che esprimono le matrici extracromosomici descritti al punto 1.2 per la ultravioletti (UV) / trimethylpsoralen (TMP), il protocollo di integrazione11. </li><li> Bleach sincronizzare gli animali 12 e coltivarli in Nematode crescita media (NGM) piastre contenenti OP50 E. coli alla fase L4. Lavare L4 animali in piatti NGM con M9 tampone per rimuovere OP50 E. coli batteri. </li><li> Rimuovere tampone M9 e aggiungere 50-100 ml di 30 mg / ml soluzione TMP lavoro sul verme pellet nel tubo 1,5 ml microcentrifuga. Avvolgere la provetta 1,5 ml in un foglio di bloccare la luce ambiente, e agitare delicatamente su un mixer per 15 minuti nella pellicola protettiva provetta 1,5 ml microcentrifuga. Trasferire i vermi nel TMP / worm soluzione ad un grande piatto NGM testa di serie. </li><li> Esporre i vermi sulla piastra NGM ai raggi UV 350 μJ (X100) utilizzando un Stratalinker 2400. Quindi aggiungere i vermi ad una piastra contenente OP50 NGM e incubare al buio per 5 ore. </li><li> Scegli 50 vermi transgenici a circa 10 piastre di teste di serie (2-5 vermi ogni piastra). Trasferimento P0 è a piatti nuovi ogni 24 ore per 3 giorni. </li><li> Scegli 250 F1 clonale unnimals (1 animale per piastra). Clone out 2-4 animali F2 da F1 ogni piatto. Controllare animali F3 per cercare una linea di trasmissione 100% cercando in (p) ofm-1 :: pattern di espressione GFP sotto un microscopio da dissezione fluorescente. </li><li> Outcross la riga finale integrato (s) con gli animali di tipo selvatico N2 5 volte. Si farà riferimento alla linea finale integrato di pyIs500, e la GFP-tagged EGL-4 molecola come GFP :: EGL-4. </li></ol> 2. La coltivazione e la manutenzione degli animali da saggi traslocazione nucleare <ol><li> Coltivate le pyIs500 animali a 25 ° C su piastre standard di 10 centimetri NGM (vedi sotto per ricetta) seminato con OP50 E. coli 13 (Figura 1). </li><li> Il giorno 1, scegliere 4-5 L4 pyIs500 animali da piastre coltivate a 25 ° C su cinque distinti 10 centimetri OP50 E. coli seminato piastre e incubare a 25 ° C (cioè, L4 4-5 animali per piastra). </li><li> Il giorno 4, lavare adulto populations di pyIs500 animali al largo delle piastre NGM di grandi dimensioni utilizzando S-basale del buffer (vedi sotto per ricetta). Utilizzare pipette in vetro usa e getta per trasferire gli animali, in quanto si attaccano al lato della puntali in plastica. </li></ol> 3. A lungo termine indotta Odore saggi traslocazione nucleare <ol><li> Lavare le pyIs500 animali 3 volte in S-basale per rimuovere i batteri. Non centrifugare animali tra un lavaggio, ma piuttosto permettere agli animali di stabilirsi per gravità in un fermo 1,5 ml microcentrifuga tubo. E &#39;fondamentale per garantire tutti i batteri viene rimosso, e che i paté NGM sono esenti da contaminazione, i batteri residui influenzerà negativamente l&#39;esito del test traslocazione. Inoltre, abbiamo trovato che la sterilizzazione in autoclave le provette da 1,5 ml microcentrifuga produce una proprietà &quot;appiccicoso&quot; sulle pareti interne del tubo che causa gli animali di aderire alle pareti, ed utilizzare quindi non autoclave provette da 1,5 ml microcentrifuga. </li><li> Mentre gli animali si stanno stabilendo tra un lavaggio, portare tegli adattamento soluzione. Aggiungere 100 ml di S-basale buffer su un cilindro graduato da 100 ml laurea. Aggiungi adattamento odore della S-basale e sigillare il cilindro con una striscia di parafilm. Se si esegue l&#39;adattamento benzaldeide aggiungere 7,5 ml di benzaldeide a 100 ml di S-Basal, per l&#39;adattamento butanone aggiungere 11 ml di 2-butaone a 100 ml di S-Basal. Capovolgere delicatamente (non agitare) il cilindro laurea contenente S-basali e l&#39;odore 30 volte per creare una emulsione uniforme. </li><li> Dopo l&#39;ultimo lavaggio in S-basale, permettere agli animali di risolvere e rimuovere tutto il liquido. Etichettare una provetta &quot;+ ve&quot; o &quot;adattare&quot; e l&#39;altro tubo &quot;-ve&quot; o &quot;unadapt&quot;. Poi aggiungere 1 ml della miscela di adattamento alla provetta adattamento (+ ve), e aggiungere 1 ml di S-basale al tubo disadattato microcentrifuga di comando (-ve). Cercate di mantenere il numero di animali tra 100 e 200 in ciascun tubo. Con una certa pratica, è possibile stimare il numero approssimativo di animali in un pellet ad occhio. </li><li> Inserire un 1,5 ml microcentrifuga tubo tappo di protezionead ogni provetta (impedisce coperchi da aperto popping) e posizionare i tubi su un rotatore per 80 min. Abbiamo trovato che l&#39;esposizione odore tra 60 min e 80 min produce risultati simili (sia risultato a lungo termine risposte di adattamento). Tuttavia, l&#39;80 minuti di esposizione offre risultati più coerenti nelle nostre mani. Quindi tutti i nostri saggi a lungo termine di adattamento sono standardizzati utilizzando un odore di esposizione 80 min (Figura 2). </li><li> Dopo 80 minuti, lavare gli animali 3 volte in S-basale. Consentire agli animali di risolvere per gravità tra ogni lavaggio. </li></ol> 4. A breve termine Odore indotte saggi traslocazione nucleare <ol><li> In caso di breve termine dell&#39;adattamento odore, utilizzare lo stesso protocollo per lungo termine saggi traslocazione odore indotte (Fasi 3,1-3,4), ma invece di incubazione delle pyIs500 animali nell&#39;adattare odori per 80 min, utilizzare una esposizione 30 min . La quantificazione di odore indotte saggi traslocazione nucleare descritta di seguito (Passaggio 5) è lo stesso per lungo e breve term esposizioni. </li></ol> 5. Segnare l&#39;Odore indotto degli eventi nucleari Traslocazione <ol><li> Fai 2% pastiglie agarosio contenenti 5 mM sodio azide (NaN 3) sciogliendo elettroforesi su gel di agarosio in dH 2 0. Mettere una goccia di agarosio fuso contenente NaN 3 su un vetrino per microscopio. Porre immediatamente un altro vetrino da microscopio di vetro sulla prima diapositiva per appiattire la goccia di agarosio fuso. Lasciare agire per 30 secondi, e poi delicatamente prendere in giro a parte la vetrini lasciando un vetrino da microscopio con un tampone piatto agarosio di circa 1 mm. </li><li> Dopo l&#39;ultimo lavaggio in S-basale, permettere ai vermi di stabilirsi nel tubo 1,5 ml microcentrifuga e rimuovere tutto il liquido. Quindi aggiungere gli animali goccia a goccia sui pad agarosio di vetrini da microscopio diversi. E &#39;importante mantenere il numero di animali per ogni diapositiva a tra 50 e 100. Troppi animali per vetrino si complicano punteggio e spesso creano un bagliore spargendo dopo blu-luce di eccitazione. Liquido in eccesso puòessere cattivi dai pad agarosio con un piccolo Kim-wipe. Collocare un bicchiere 0,5 millimetri copertura antiscivolo sul tampone e lasciare riposare per 2 min per consentire l&#39;NaN 3 per immobilizzare gli animali. Nota, gli animali possono essere recuperato presso le diapositive dopo aver segnato. Gli animali si riprenderà dal 3 NaN anestetico dopo ~ 1 ora in caso di cessione di una piastra NGM seminato in una goccia di tampone M9. </li><li> A questo punto è indispensabile che ogni sperimentatore elaborare una chiave per tenere traccia che scorrono è sperimentale (+ ve) diapositiva e che scorrono è il controllo (-ve) slitta, e passare le diapositive ad un&#39;altra persona che ciecamente segnerà il GFP: EGL-4 modello di localizzazione. Ciò permette di controllare per ogni pregiudizio sperimentatore. </li><li> Punteggio compreso tra 50 e 100 capi per vetrino utilizzando un microscopio a fluorescenza in posizione verticale con gli obiettivi di ingrandimento 10X, 40X e 63X e GFP / RFP filtri. In primo luogo, individuare gli animali a 10 ingrandimenti con campo chiaro illuminazione. In secondo luogo, individuare il neurone AWC spegnendo il bright-campo di illuminazione e utilizzando il filtro RFP. I (p) ODR-1 :: unità DsRed espressione in AWB e neuroni AWC. I neuroni hanno AWC distintivi corpi cellulari di forma ovale rispetto AWB corpi cellulari che sono più piccoli e di forma circolare (Figura 2). </li><li> Una volta che il corpo cellulare AWC è stato localizzato, passare al filtro GFP e registrare la localizzazione di GFP: EGL-4 come nucleare o citoplasmatico. Utilizzando un contatore permette di mantenere lo sperimentatore guardare nell&#39;oculare mentre segnando la diapositiva. Nota: 5,1-5,5 passaggi vengono ripetuti almeno 3 volte in giorni diversi per ottenere dati statisticamente significativi (figure 1-2). </li></ol> 6. Il monitoraggio GFP-tagged EGL-4 durante il recupero da lungo termine adattamento Odore <ol><li> Passo dopo 3,5, dividere le popolazioni di animali pyIs500 in aliquote multiple e punteggio per GFP nucleare rispetto citoplasmatica :: EGL-4 nel AWC punto al tempo zero (cioè, dopo 80 minuti di esposizione) ed multiple punti temporali successivi (Figura 3). </li><li> Trasferire sia il controllo (-ve) e sperimentali (+ ve) animali con pipette in vetro usa e getta su piastre NGM. </li><li> Consentire agli animali di recuperare per varie lunghezze di tempo e di riesaminare la localizzazione di GFP :: EGL-4in AWC ad ogni tempo, come nei passaggi 5,1-5,5. </li></ol> 7. Materiale Piastre NGM: Per 1 L aggiungere a 1 L DDH 2 0: 21 g Bacto-agar, 3 g Cloruro di sodio (NaCl), 2,5 g Bacto-peptone. Autoclave Media. Dopo la sterilizzazione in autoclave, raffreddare l&#39;agar fino a quando la temperatura indica 54 ° C. Aggiungere le seguenti soluzioni: 25 ml 1M tampone fosfato di potassio, 1 ml 1 M di solfato di magnesio (MgSO4), 1 ml 1 M di cloruro di calcio (CaCl 2); 1 colesterolo ml in etanolo (5 mg / ml di brodo). 1 M bibasico K 2 HPO 4 174,2 g / mol: Per 500 ml: In 400 ml DDH 2 O, sciogliere 87,1 g Dibasic K 2 HPO 4. Regolare il volume a 500 L con DDH 2 O. Filtrare sterilizzare con un 500 ml Unità di filtro sterile. 1 M monobasico di KH 2 PO 4 136,1 g / mol: Per 1 L: In 800 ml DDH 2 O, sciogliere 136,1 g monobasico di KH 2 PO 4. Regolare il volume a 1 L con DDH 2 O. Filtrare sterilizzare con un 500 ml Unità di filtro sterile. 1 M fosfato di potassio (K 3 PO 4) pH 6.0: Per 1 L: In una unità 1 L filtro sterile, aggiungere 868 ml di 1 M monobasico di KH 2 PO 4. Permettere all&#39;unità di filtrare, poi aggiungere 132 ml di 1 M bibasico K 2 HPO 4. Permettere all&#39;unità di filtrare tutto il liquido. Rimuovere l&#39;unità di filtro e aggiustare il pH a 6,0. Conservare a temperatura ambiente. S-basale: Per 1 L: aggiungere 50 ml 1M K 3 PO 4 tampone, pH ~ 6.0, e 20 ml di NaCl 5M aq. Riempire a 1 L conDDH 2 O. Autoclave soluzione. M9: Per 1 L: 3 g KH 2 PO 4, 6 g di Na 2 HPO 4, 5 g NaCl, 1 ml 1 M MgSO 4, H 2 O. Riempire a 1 L con DDH 2 0. Sterilizzare in autoclave.

<ol>
	<li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14 (4), 803 (1995).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+modulate+olfactory+acuity,+discrimination,+and+memory+in+Caenorhabditis+elegans.">Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.</a> Learning and Memory. 4 (2), 179 (1997).</li><li>Bargmann, C. I., 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=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Neuron. 74 (3), 515 (1993).</li><li>L'Etoile, N. D., 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+cyclic+GMP-dependent+protein+kinase+EGL-4+regulates+olfactory+adaptation+in+C.+elegans.">The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans.</a> Neuron. 36, 1079-10 (2002).</li><li>Lee, J. I., 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=Nuclear+entry+of+a+cGMP-dependent+kinase+converts+transient+into+long-lasting+olfactory+adaptation.">Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.</a> PNAS. 107 (13), 6016 (2010).</li><li>O'Halloran, D. M., 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=Regulators+of+AWC-mediated+olfactory+plasticity+in+Caenorhabditis+elegans.">Regulators of AWC-mediated olfactory plasticity in Caenorhabditis elegans.</a> PLoS Genetics. 5 (12), e1000761 (2009).</li><li>O'Halloran, D. M., 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=Changes+in+cGMP+levels+affect+the+localization+of+EGL-4+in+AWC+in+Caenorhabditis+elegans.">Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans.</a> PLoS ONE. 7 (2), e31614 (2012).</li><li>Mello, C. C., 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=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO Journal. 10, 3959-39 (1991).</li><li>Miyabayashi, T., 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=Expression+and+function+of+members+of+a+divergent+nuclear+receptor+family+in+Caenorhabditis+elegans.">Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans.</a> Dev. Biol. 215, 314 (1999).</li><li>L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+and+odor+discrimination+are+mediated+by+the+C.+elegans+guanylyl+cyclase+ODR-1.">Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1.</a> Neuron. 25 (3), 575 (2000).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451 (1995).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> Wormbook. , (2006).</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 (1), 71 (1974).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <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+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+Dopamine+Neuron+Degeneration+Assay+for+the+Validation+of+Potential+Parkinson's+Disease+Genes.">Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes.</a> J. Vis. Exp. (17), e835 (2008).</li><li>Pittenger, C., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+general+mechanisms+for+long-lasting+plasticity:+Aplysia+and+the+hippocampus.">In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358 (1432), 757 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;odore indotta ingresso nucleare di GFP-tagged EGL-4 molecola qui descritta fornisce una lettura solida molecolare di adattamento olfattivo in C. elegans. L&#39;odore indotte saggi traslocazione nucleare sono semplici e richiedono solo pochi giorni di tempo di preparazione. Il pyIs500 animale che abbiamo costruito per questi test, esprime un marcatore che illumina il neurone AWC oltre ad esprimere la GFP-tagged EGL-4 proteine. Pertanto, riteniamo che uno sperimentatore con poca o nessuna esperienz...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Bacto Agar </td><td> Difco </td><td> DF0140-07-4 </td><td> NGM piastre </td></tr><tr><td> Cloruro di sodio </td><td> Fisher Chemical </td><td> S671-10 </td><td> NGM piastre </td></tr><tr><td> Bacto Peptone </td><td> Difco </td><td> DF0118-07-2 </td><td> NGM piastre </td></tr><tr><td> Fosfato di potassio bibasico </td><td> Fisher Chemical </td><td> S375-500 </td><td> S-Basal buffer e piastre NGM </td></tr><tr><td> Potassio fosfato monobasico </td><td> Fisher Chemical </td><td> P285-500 </td><td> S-Basal buffer e piastre NGM </td></tr><tr><td> Kimwipes - Piccolo </td><td> Kimberly-Clark </td><td> LS2770 </td><td></td></tr><tr><td> Etanolo 100% </td><td> Gold Shield chimicaCo. </td><td> 43196-115 </td><td> odori diluizione per saggi di chemiotassi </td></tr><tr><td> Cloruro di calcio </td><td> Sigma-Aldrich </td><td> C8106-500G </td><td> NGM piastre </td></tr><tr><td> Magnesio solfato </td><td> MP Biomedicals </td><td> 150136-500G </td><td> NGM piastre </td></tr><tr><td> Sodio azide 99% </td><td> Fisher Scientific </td><td> ICN10289180 </td><td> Anestetico </td></tr><tr><td> Agarosio - ultrapura </td><td> Invitrogen </td><td> 16500-500 </td><td> Agarosio pastiglie </td></tr><tr><td> Benzaldeide </td><td> Sigma-Aldrich </td><td> B1334-100G </td><td> Odore AWC </td></tr><tr><td> Butanone, ACS grado </td><td> Sigma-Aldrich </td><td> 360473-500ML </td><td> Odore AWC </td></tr><tr><td> Tubi - microcentrifuga 1,5 ml colorati </td><td> Denville </td><td> LS8147 </td><td></td></tr><tr><td> Pipettare Pasteur monouso in vetro 5-3/4 &quot; </td><td> Fisher Scientific </td&gt; <td> 13-678-20B </td><td></td></tr><tr><td> Stratalinker </td><td> Stratagene </td><td> Stratalinker 2400 </td><td> Integrazione UV </td></tr><tr><td> Filtrare sotto vuoto bottiglia - 500 ml </td><td> Nalgene </td><td> 09-740-25B </td><td></td></tr></tbody></table>

a molecular readout of long-term olfactory adaptation in c. elegans

Durante la stimolazione prolungata più neuroni sensoriali si adatterà la loro risposta diminuendo la loro sensibilità al segnale. La risposta di adattamento aiuta attenzione la forma e protegge anche le cellule provenienti da oltre-stimolazione. Adattamento nel circuito olfattivo di C. elegans è stato descritto da Colbert e Bargmann 1,2. Qui, gli autori hanno definito i parametri del paradigma adattamento olfattivo, che hanno usato per la progettazione di uno screening genetico di isolare mutanti difettosi nella loro capacità di adattarsi agli odori volatili rilevati dalle Amphid cellule alari di tipo C (AWC) neuroni sensoriali. Quando tipo selvatico C. elegans animali sono esposti a un interessante AWC-sentito odore 3 per 30 minuti che si adatterà la rispondenza alle l&#39;odore e quindi ignorare l&#39;odore adattando in un saggio di chemiotassi comportamentale per ~ 1 ora. Quando tipo selvatico C. elegans animali sono esposti a un interessante AWC-sentito odore di ~ 1 ora saranno quindi ignorare l&#39;odore di adattare di achemotaxis test comportamentali per circa 3 ore. Queste due fasi di adattamento olfattivo in C. elegans sono stati descritti come breve termine l&#39;adattamento olfattivo (indotta dopo 30 minuti di esposizione odore), ea lungo termine adattamento olfattivo (indotto dopo 60 minuti di esposizione odore). Più tardi lavorare da L&#39;Etoile et al., 4 scoperto una proteina chinasi G (PKG) chiamato EGL-4 che è necessario sia per l&#39;adattamento a breve e lungo termine nei neuroni olfattivo AWC. Il-4 EGL proteina contiene una sequenza di localizzazione nucleare che è necessario a lungo termine risposte di adattamento olfattive, ma non indispensabili per il breve termine, le risposte di adattamento olfattivo nel AWC 4. Taggando EGL-4 con una proteina fluorescente verde, è stato possibile visualizzare la localizzazione di EGL-4 nel AWC durante l&#39;esposizione prolungata odore. L&#39;utilizzo di questo completamente funzionale GFP-tagged EGL-4 (GFP :: EGL-4) molecola siamo stati in grado di sviluppare una lettura molecolare di lungo termine adattamento olfattivo nel AWC 5. Utilizzando questo mlettura olecular di adattamento olfattivo siamo stati in grado di eseguire entrambi gli schermi in avanti e all&#39;indietro genetici per identificare gli animali mutanti che presentano i modelli difettosi localizzazione subcellulare di GFP :: EGL-4 nel AWC 6,7. Qui si descrive: 1) la costruzione di GFP :: EGL-4 animali che esprimono, 2) il protocollo per la coltivazione di animali a lungo termine saggi traslocazione odore indotta nucleari, e 3) il punteggio di lungo termine odore indotta traslocazione evento nucleare e il recupero (ri-sensibilizzazione) dal nucleare GFP :: EGL-4 Stato.

Un esempio del modello di localizzazione GFP :: EGL-4in l&#39;AWC prima e dopo esposizione prolungata odore è mostrato in Figura 2. Prima dell&#39;esposizione prolungata odore, GFP :: EGL-4 è localizzato al citosol della AWC (Figura 2B), e dopo 80 minuti di esposizione GFP odore :: EGL-4 è localizzata nel nucleo della AWC (Figura 2D). A livello comportamentale, gli animali con citosolica GFP :: EGL-4 in AWC sono attratti da una sorgente puntiforme di odore (F...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Qui descriviamo una lettura molecolare di lungo termine dell&#39;adattamento olfattivo<em&gt; Caenorhabditis elegans</em&gt;. La proteina chinasi G, EGL-4, è necessario per le risposte di adattamento stabili nella coppia primaria neurone sensoriale chiamato AWC. Durante prolungati odore di esposizione EGL-4 trasloca dal citosol al nucleo della AWC.

Una lettura molecolare di lungo termine adattamento olfattivo nel<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

a-molecular-readout-of-long-term-olfactory-adaptation-in-c-elegans

Research

JoVE Journal

Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

10.7K Views.  George Washington University. Qui descriviamo una lettura molecolare di lungo termine dell'adattamento olfattivo Caenorhabditis elegans. La proteina chinasi G, EGL-4, è necessario per le risposte di adattamento stabili nella coppia primaria neurone sensoriale chiamato AWC. Durante prolungati odore di esposizione EGL-4 trasloca dal citosol al nucleo della AWC.

Video: A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Learning Positivo Butanone, a breve termine e lungo termine saggi di memoria associativa

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Ricerca

Neuroscienze

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo Laser assotomia in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Quantificazione automatica di fluorescenza in Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

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, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

A lungo termine comportamentale inseguimento di Liberamente Nuoto debolmente elettrico Pesce

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

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 ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Analisi automatizzata di<em&gt; C. elegans</em&gt; Comportamento Swim Utilizzando Celest Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Quadrupla Immunostaining della lampadina olfattiva per la visualizzazione di codici di identificazione molecolare Axon sensoriale olfattiva

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Tramite un Chip olfattiva di microfluidica adattato per l'Imaging dell'attività neuronale in risposta ai feromoni in neuroni di testa maschio C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generazione e manutenzione a lungo termine di nervi liberi<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...