Vorremmo ringraziare i membri attuali e precedenti del laboratorio link che ha contribuito a sviluppare i protocolli descritti qui, in particolare Gin Fonte, che ha sviluppato il procedimento originale. Questo lavoro è stato supportato da riconoscimenti dal NIH (AG12423) e l&#39;Alzheimer Association (Premio Zenith) alla Cdl

1) Preparazione dei nematodi media di crescita piastre per test paralisi. Saggi di paralisi vengono eseguite sullo standard nematode crescita Media (NGM) piastre solitamente usata per C. elegans propagazione e genetica (Stiernagle, 2006). <ol><li> Autoclave NGM la soluzione contenente NaCl, Agar e Peptone in una beuta e aggiungere la quantità appropriata di sterili CaCl 2, uracile, colesterolo, 1M MgSO 4 e 1M KPO 4. Aliquota 10 ml di liquido in NGM ogni mm 60 x 15 mm piastra di Petri. Lasciare NGM a solidificare notte a temperatura ambiente. </li><li> Se il test per gli effetti dei composti / estratti, aliquota dell&#39;estratto su ogni piatto e utilizzare una spatola per distribuire uniformemente l&#39;estratto sulla superficie della piastra. Lasciare che l&#39;estratto secco di notte a temperatura ambiente. Volumi oltre 1 ml richiederà più tempo per asciugare. </li><li> Spot ogni piatto con 250 ml di E. coli ceppo OP5O cresciuto nel Media LB durante la notte per una densità ottica di 0,4-0,6. Permettono ai batteri di asciugare durante la notte a temperatura ambiente. </li></ol> Variabili rilevanti L&#39;età delle lastre ha un effetto significativo sul saggio paralisi, come vecchi piatti tendono ad asciugarsi. Usare piatti versato entro una settimana saggio paralisi. Per un risultato ideale, versare piastre 3-4 giorni prima dell&#39;inizio delle popolazioni verme sincrono [vedi (2) qui sotto]. Per ogni esperimento, utilizzare solo piatti versato dalla stessa partita. Alterare le dimensioni del prato (cioè macchiato vs diffusione) e / o il tipo di batteri anche alterare il comportamento del worm. Saggi di paralisi può essere effettuata con metodi alternativi E. coli come fonte di cibo (per esempio, l&#39;HT115 ceppo usato per l&#39;alimentazione degli esperimenti di RNAi), ma questo può cambiare la cinetica di paralisi, il che richiede adeguati controlli interni. 2) Preparazione di Age-sincrono popolazioni Worm Ceppo CL4176 (SMG-1 (cc546 ts) I; dvIs27 [myo-3 / Aβ ​​minigene + rol-6 (su1006) gene marcatore] X è più comunemente usato per il saggio Aβ indotta dalla paralisi Questo ceppo e altri modelli transgenici sono disponibili. ai ricercatori universitari per una tassa nominale dalla Genetica Caenorhabditis Center (http://www.cbs.umn.edu/CGC/). <ol><li> Per massimizzare la sincronia età della popolazione sottoposta a test è preferibile iniziare con una generazione sincronizzato dei genitori. Una settimana prima di iniziare l&#39;analisi della paralisi della popolazione prova, utilizzare un selettore a vite senza fine di platino per trasferire 20-30 adulti gravido su 10 piatti diversi NGM centimetri diffuso con OP50 per un uovo 2 ora giaceva a 16 ° C (la temperatura permissiva per CL4176 ). Rimuovere gli adulti gravido e consentire la progenie di crescere per 7 giorni, quando ormai saranno &quot;secondo giorno&quot; adulti gravido. </li><li> (Giorno 1) Gli adulti secondo giorno gravido vengono utilizzati per preparare l&#39;età sincrono popolazioni di prova. Per ogni condizione sperimentale, l&#39;oggetto è quello di generare piastre triplice copia contenente 50-75 anni-sincrono vermi. Utilizzando un selettore verme platino, il trasferimento 10-12 del 2 adulti giorno gravido su 60 millimetri (10 ml) piastre NGM macchiato con OP50. Lasciare che il worm per deporre le uova a 16 ° C per 2 ore, poi prendere via gli adulti e consentire uova a schiudersi e crescere a 16 ° C. A questo punto è meglio avere qualcuno che non sarà segnare le targhe di codice loro, in modo che il marcatore sarà cieco alle condizioni sperimentali. </li></ol> Variabili rilevanti La fase di sviluppo embrionale, quando uova vengono deposte (~ i 26-cell stadio per i vermi wild-type in condizioni ottimali) è una funzione dell&#39;età adulta e della nutrizione. Se gli adulti gravido utilizzati per la preparazione della popolazione di età superiore o test sono affamati, uova fase successiva saranno stabilite, riducendo la sincronia età della popolazione e la riproducibilità della cinetica paralisi. E &#39;essenziale che mono-axenic (cioè, solo OP50 batteri) le culture sono utilizzati (contaminanti microbici può seriamente influire su questo test), e la riproducibilità è più alta se le piastre tre esemplari hanno un numero simile di vermi. 3) Transgene induzione e analisi Paralisi <ol><li> (Giorno 3) piastre cambio marce alte a 25 ° C 48 ore dopo la fine dell &#39;uovo sincrono laici; worm dovrebbe essere al terzo stadio larvale (L3). Piastre devono essere disposte senza accatastamento in un incubatore a 25 ° C per consentire tutte le lastre di raggiungere 25 ° C, allo stesso tempo. </li><li> (Giorno 4) Iniziare segnando la paralisi di vermi (ceppo CL4176) a 18-20 ore dopo l&#39;inizio del cambio marcia. A questo punto, tutti i vermi nella popolazione dovrebbe avere raggiunto la larvale quarto (L4) stadio. Continuare ad ottenere in due ore con incrementi fino a che tutti i vermi su ciascuno dei piatti sono paralizzati. Per ragioni sconosciute, la paralisi non è nemmeno tutta la lunghezza del corpo: la regione della testa è l&#39;ultima parte del worm di cessare in movimento. Così, wORM che hanno recentemente avviato la paralisi non può tradurre in tutto il piatto, ma può muovere la testa, aprendo i batteri intorno al loro anteriore e lasciando un &quot;alone&quot; di batteri cancellata. Questi worm inevitabilmente diventerà completamente paralizzato, e quindi worm con aloni sono classificati come paralizzati. Alcuni worm non avrà aloni, ma non anche mostrare movimento spontaneo: queste sono testati da incitando con il selettore worm. Se un worm pungolati non può subire una completa propagazione delle onde su sollecitazione del corpo, è anche notato come paralizzato. Per il punteggio efficiente, è più facile spostare il worm paralizzato per un settore senza macchia del piatto in modo che non sarà involontariamente rescored del prossimo periodo di punteggio. (Questo permette anche mis-classificati worm per dimostrare il movimento, consentendo una correzione di punteggio). </li><li> Per generare una curva di paralisi, in ogni fase la frazione di vermi su ogni piatto che non sono stati paralizzati viene convertito in una percentuale, e la media percentuale &quot;unparalyzed&quot; è rilevata in tempo di iniziazione salire di marcia. (Il razionale per la stampa in questo modo è che la curva risultante è formalmente analoga a una curva di sopravvivenza, e quindi possono essere analizzati utilizzando le normali non parametrica statistiche di sopravvivenza.) </li></ol> Variabili rilevanti Il cambio marcia di vermi myo-3/Aβ transgeniche deve avvenire prima che raggiungano la metà L4 fase di paralisi, che sarà avviata, in quanto il promotore myo-3 sia maturo e regolata, e ha notevolmente ridotto espressione in L4 in ritardo o vermi adulti. Allo stesso modo, l&#39;espressione di questo transgene è bloccato se i vermi diventano affamati, quindi è essenziale che i vermi sono ben nutriti per tutto l&#39;esperimento. Non abbiamo mai osservato un worm paralizzato per recuperare in qualsiasi condizione, e dopo 12-16 ore di salire di marcia, tutti CL4176 vermi paralizzare, anche se sono downshifted a 16 ° C. Nel ceppo CL4176, sovraregolazione sufficiente di espressione del transgene a causare paralisi può avvenire a temperature più basse (ad esempio, 20-25 ° C), anche se i tempi di paralisi sarà alterato. Il tasso di paralisi dipende dal ceppo specifico transgenici utilizzati (che abbiamo costruito ceppi myo-3/Aβ sia con i tassi più veloce e più lento rispetto CL4176), quindi con ceppi alternativa alla finestra di punteggio potrebbe essere necessario un aggiustamento. 4) Rappresentante Risultati Mostrato nella Figura 1 è un grafico di una curva di paralisi per CL4176, sia non trattati ed esposti a 6,27 mM caffeina (concentrazione finale in lamiera NGM). Il risultato è un trattamento in un ritardo statisticamente molto significativo nella paralisi di circa 4 ore, che noi interpretiamo come indicativi di soppressione di tossicità Aβ in questo modello. Mostrato nella Figura 2 è un esperimento indipendente saggiare gli effetti di Cafestol, un altro composto trovato nel caffè. Questo grafico è tipico per i trattamenti che non hanno impatto sulla tossicità Aβ. Si noti che in entrambi gli esperimenti circa la metà della popolazione non trattata CL4176 sono paralizzati a 24 ore, e la paralisi è completa di 28 ore. Questi sono tempi tipici per la paralisi del controllo CL4176 popolazioni. Deviazioni significative da questo momento sono indicativi di mutate condizioni ambientali o l&#39;eliminazione spontanea di copie del transgene. (CL4176 contiene un cromosomicamente integrato serie transgenici con più copie del transgene myo-3/Aβ. Sebbene questo array transgenico è normalmente abbastanza stabile, la diffusione del ceppo a temperature&gt; 16 ° C selezionerà per i vermi rari che hanno subito un l&#39;eliminazione spontanea di transgeni, con conseguente varianti che hanno ridotto l&#39;espressione Aβ e dei risultati paralisi più lento.) <img src="/files/ftp_upload/2252/2252fig1.jpg" alt="Figura 1" /> Figura 1. Curva paralisi dei ceppi CL4176 vermi non trattati o esposti a 6,27 caffeina mM (Sigma). I tempi indicati sono da inizio del cambio marcia temperatura. Barre di errore = SEM <img src="/files/ftp_upload/2252/2252fig2.jpg" alt="Figura 2" /> Figura 2. Curva di paralisi per CL4176 vermi esposti a 0,48 mM Cafestol (Sigma). Barre di errore = SEM

<ol>
	<li>Arya, U., Dwivedi, H., Subramaniam, 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=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</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. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo che abbiamo descritto può essere efficacemente utilizzato per test gli effetti dei composti sulla tossicità Aβ. Esempi pubblicati includono gli effetti costituenti dell&#39;estratto di Ginkgo biloba (Wu et al. 2006) e reserpina (Arya et al. 2009). Composti che sono stati protettivo contro la tossicità Aβ in altri sistemi hanno anche dimostrato di essere protettivo in questo modello (ad esempio, la memantina, i nostri risultati non pubblicati). Una logica importante per stabilire questo modello è che gli strumenti genetici e molecolari ben sviluppato in C. elegans può essere usato per indagare i meccanismi di tossicità Aβ. Quindi, le mutazioni effetti specifici sulla tossicità Aβ può essere analizzati (ad esempio, l&#39;effetto di ridurre insulino-simile di segnalazione introducendo un daf-2 perdita di funzione di mutazione, Florez-McClure et al. 2007), e questo modello può anche essere usato per esaminare gli effetti di sovra-espressione transgeni (Fonte et al. 2008). Di recente abbiamo fatto ampio uso di questo modello per esaminare gli effetti sulla tossicità di singole sostituzioni aminoacidiche nella Aβ (dati non pubblicati). Il modello transgenico qui descritto analisi l&#39;effetto di acuta espressione Aβ sulla funzione delle cellule muscolari. Al tempo delle cellule muscolari e paralisi loro sarcomero sono intatte, il fenotipo paralisi non è il risultato della morte delle cellule muscolari. Tuttavia, dopo la paralisi (~ 48 ore dopo il cambio marcia iniziale) vi è una ripartizione di integrità muscolare e la morte dei vermi. Non abbiamo studiato questo effetto a valle di espressione Aβ o utilizzati come marker di tossicità. La Aβ modello inducibile espressione qui descritto non è appropriato per indagare trattamenti che modulano β-amiloide formazione di per sé. Anche se i vermi transgenici con la forma costitutiva espressione Aβ depositi di amiloide facilmente rilevabili (Fay et al 1998;.. Link et al 2001), vermi transgenici con espressione inducibile Aβ raramente hanno depositi di amiloide e il fenotipo paralisi appare indipendente dalla deposizione di amiloide (Drake et al. 2003). I nostri risultati sono coerenti con la visione che la tossicità acuta dei risultati indotta da Aβ espressione l&#39;accumulo di Aβ solubile oligomerici, non deposizione di amiloide.

<table border="1"><tbody><tr><td> Reagenti </td><td> Quantity/2L </td><td> Azienda / Catalogo # </td><td> Commenti </td></tr><tr><td> NaCl </td><td> 6g </td><td> Sigma-Aldrich / S9888 </td><td></td></tr><tr><td> Agar </td><td> 40g </td><td> Sigma-Aldrich / A7002 </td><td></td></tr><tr><td> Peptone </td><td> 5g </td><td> Sigma-Aldrich / P0556 </td><td></td></tr><tr><td> DDH 2 O </td><td> 2L </td><td></td><td></td></tr><tr><td> 1M CaCl 2 </td><td> 2 ml </td><td></td><td> 147.02mg/mL in DDH 2 O </td></tr><tr><td> Uracile </td><td> 2 ml </td><td></td><td> 2mg/ml in DDH 2 O </td></tr><tr><td> Colesterolo </td><td> 2 ml </td><td></td><td> 5mg/ml in etanolo (non in autoclave) </td></tr><tr><td> 1M MgSO 4 </td><td> 2 ml </td><td></td><td> 246.5mg/mL in DDH 2 O </td></tr><tr><td> 1M KPO 4 </td><td> 50mL </td><td></td><td> 98mg KH 2 PO 4. ML, 48mg K2HPO4/mL in DDH 2 O, pH </td></tr><tr><td> Erlenmeyer Flask </td><td></td><td> VWR </td><td></td></tr></tbody></table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

Accumulo del peptide β-amiloide (Aβ) è generalmente ritenuto centrale per l&#39;induzione della malattia di Alzheimer, ma il meccanismo in questione (s) di tossicità sono ancora poco chiari. Aβ è depositato per via intramuscolare nella miosite inclusione dell&#39;organo, un grave miopatia umano. Il intensamente studiato verme nematode Caenorhabditis elegans può essere transgenically progettato per esprimere Aβ umano. A seconda del tessuto o la tempistica di espressione Aβ, vermi transgenici possono avere fenotipi facilmente misurabili che servono come lettura di tossicità Aβ. Per esempio, vermi transgenici con pan-neuronale espressione Aβ avere difetti è l&#39;apprendimento associativo (Dosanjh et al 2009.), Mentre i vermi transgenici con costitutiva muscolo-specifica espressione mostrano un progressivo, età-dipendente fenotipo paralisi (Link, 1995; Cohen et al . 2006). Uno particolarmente utile C. elegans modello si avvale di un sensibile alla temperatura mutazione del sistema di sorveglianza di mRNA ingegnere temperatura-inducibile espressione muscolare di un transgene Aβ, con conseguente paralisi fenotipo riproducibile su cambio marcia temperatura (Link et al. 2003). Trattamenti che contrastare la tossicità Aβ in questo modello [per esempio, espressione di un transgene di protezione (Hassan et al. 2009) o l&#39;esposizione a estratti di Ginkgo biloba (Wu et al. 2006)] riproducibile alterare il tasso di paralisi indotta dalla temperatura salire di marcia di questi transgenici vermi. Qui si descrive il nostro protocollo per misurare il tasso di paralisi in questo C. transgenico elegans modello, con particolare attenzione alle variabili sperimentali che possono influenzare questa misura.

Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

Il verme nematode intensamente studiato<em&gt; Caenorhabditis elegans</em&gt; Può essere transgenically progettato per esprimere l&#39;umano peptide β-amiloide (Aβ). Espressione indotta di Aβ in<em&gt; C. elegans</emMuscolare&gt; porta ad un rapido, fenotipo paralisi riproducibile che può essere utilizzato per monitorare i trattamenti che modulano la tossicità Aβ.

Saggio β-amiloide tossicità utilizzando un transgenici C. elegans Modello

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

assaying-amyloid-toxicity-using-a-transgenic-c-elegans-model

Research

JoVE Journal

Neuroscience

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

25.3K Views.  University of Colorado. Il verme nematode intensamente studiato Caenorhabditis elegans Può essere transgenically progettato per esprimere l'umano peptide β-amiloide (Aβ). Espressione indotta di Aβ in C. elegans porta ad un rapido, fenotipo paralisi riproducibile che può essere utilizzato per monitorare i trattamenti che modulano la tossicità Aβ.

Video: Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Saggio attività locomotoria per studiare ritmi circadiani del sonno e parametri in Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

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 ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Monitoraggio e valutazione del comportamento

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

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

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

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

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the 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 ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Il saggio danneggiare il DNA in neuroni ippocampali Utilizzo del Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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, ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Sistema modello per Amylopathy Studio

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizzazione neuroblasti Citocinesi Durante<em&gt; C. elegans</em&gt; Embriogenesi

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling 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 stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

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 ...