<p class="jove_content"&gt; Ringraziamo il Dott. Shuang Liu per la lettura critica del manoscritto. La PCL12 costrutto è stato un dono forma Dr. Christopher D. Link. Questo lavoro è stato sostenuto dal National Science Foundation di due borse di studio (0842708 e 1026958) e una sovvenzione di AHA (09GRNT2250529) per FS.</p

1. Costruzione di Worms transgenici <ol><li> Trasformazione. <ol><li> Preparare pad iniezione. Mettere una goccia di caldo, 2% agarosio sciolto in acqua, su un vetrino di vetro. Introdurre rapidamente un secondo vetrino sulla goccia e toccare leggermente lo. Dopo l&#39;agarosio si è solidificato, vetrini di diapositive a parte, e cuocere il coprioggetto-pad in un forno sotto vuoto a 80 ° CO / N. </li><li> Tirare pipette. Usiamo un Sutter P-97 estrattore di tirare 1/0.5 mm OD / ID borosilicato capillari con filamento. Pipettes sono forgiati con punta chiusa che è rotto aperto in una fase successiva. </li><li> Preparare mix di iniezione. Rendere la miscela iniettabile contenente il DNA di interesse (20 ng / pl per costrutto) più DNA plasmide vuoto ad una concentrazione finale di 150 ng / ml. Centrifugare la miscela di iniezione per rimuovere qualsiasi contaminante. Questo passaggio è fondamentale perché contaminanti riducono l&#39;efficienza di trasformazione. Trasferire la top 5 microlitri (sporco e altri residui raccolgono sul fondo) in una nuova provetta per be usato in iniezione. </li><li> Caricare mix iniezione per capillarità. La parte inferiore della pipetta è immerso nella miscela di iniezione. Tipicamente 0,5 ml sono sufficienti per iniettare 100 vermi. </li><li> Caricare pipetta di iniezione. Inserire la pipetta sul supporto del micromanipolatore e romperlo aperto sfregando la punta contro il bordo di un vetrino montato su un vetrino o alternativamente contro detriti sul pad agarosio. Questo è un passo fondamentale in quanto troppo grande danno consigli del worm e troppo piccoli suggerimenti sono facilmente ostruiti. La qualità della punta rotta può essere giudicata dalla forma e soprattutto per la portata. La portata può essere valutato dalla dimensione delle bolle che scorre dalla punta aperta immersi in una goccia di olio 700 halocarbon in risposta ad una pressione di iniezione. </li><li> Trasferire i vermi a pad iniezione. Mettere una goccia di olio di 700 halocarbon su un tappetino di iniezione e trasferire più (1 o 2 per i principianti) vermi per l&#39;olio. Utilizzare un verme scegliere di spingere i vermi giù sul pad fino a quando ily aderire alla agarosio. Vermi devono essere orientati in righe con lati ventrali rivolte nella stessa direzione. Evitare di toccare la testa del verme. Se dopo vari tentativi i vermi non riescono ad aderire al pad, sostituire con un tampone fresco o aumentare agarosio spessore e / o la concentrazione. </li><li> Inserire la pipetta nel verme. Spostando la fase, posizionare il verme sotto la pipetta. Posizionare la pipetta nel nucleo centrale della gonade perché il suo citoplasma è condivisa da molti nuclei delle cellule germinali. Questo aumenta la probabilità di consegnare DNA iniettato a molti progenie. La pipetta deve trovarsi quasi parallela alla vite senza fine (~ 15 ° -25 °). Spingere verticalmente la punta della pipetta lungo il corpo del verme finché la pelle è premuto. Quindi toccare delicatamente sul manipolatore di indurre l&#39;inserimento della punta. Per ottenere i migliori risultati di iniettare due gonadi. </li><li> Iniettare la soluzione di DNA. Applicare una pressione per la pipetta fino a quando le si gonfia gonade. Arrestare il flusso e tirare il verme al largo della pipetta. Testare l&#39;ago per il flusso e quindi spostareal prossimo verme e gradini iniezioni ripetute. </li><li> Recuperare i vermi: Aggiungere una goccia (~ 10 ml) di tampone di recupero sui vermi e incubare fino a quando i vermi cominciano nuoto. Aggiungere un uguale volume di M9, attendere vermi riprendere il nuoto e ripetere più volte fino a quando la soluzione è per lo più M9. Individualmente trasferire i vermi a piastre seminate. </li></ol></li><li> Integrazione. <ol><li> Mettere 40 sano, ben nutrito, L4 vermi in un piatto fresco </li><li> Irradiate con γ-ray con 4.000 rad per 40 min. Trasferire vermi irradiati (P0) in OP50 piatti freschi, testa di serie (4 vermi / piastra). I worm possono in alternativa essere irradiati con una dose di 300 J / m 2 UV. </li><li> Trasferire 10-20 trasformanti F1 da ciascun piatto per piatti individuali, etichettare le piastre con il corrispondente P0 origine. </li><li> Singolo fuori 2-4 trasformanti F2 per ogni F1 per piatti separati. Controllare la progenie F3 per la trasmissione 100% del marcatore trasformazione. Tipicamente, 1-3% di progenie da un worm wil irradiatoDevo un evento di integrazione. </li><li> Fare scorte di tre o più linee trasformanti indipendenti. </li></ol></li></ol> 2. Saggi comportamentali <ol><li> Age-sincronizzazione. <ol><li> Crescere vermi nello standard 10 centimetri piastre NGM + OP50 E. coli fino ad una vasta popolazione di adulti gravide viene raggiunto (3-5 giorni). </li><li> Raccogliere i vermi in 50 ml provette Falcon da loro sospensione in 1 ml di tampone M9. </li><li> Aggiungere 5 ml di tampone M9 e centrifugare a 450 xg per 3 min. Scartare il surnatante. Ripetere 3-4x. </li><li> Alla fine dell&#39;ultima centrifugazione, rimuovere il surnatante e aggiungere 10 volumi di soluzione di ipoclorito di base (0,5 M NaOH, 1% ipoclorito appena miscelato) per i vermi pellet. Incubare a temperatura ambiente per circa 10 minuti. La reazione può essere monitorato lisi mettendo una goccia della reazione lisi su un vetrino ed esaminando i vermi sotto un microscopio. Quando circa il 80% dei vermi sono rotti la reazione deve essere interrotto. </li><li> Arrestare la reazione di lisi da adding lo stesso volume di tampone sterile uovo. </li><li> Raccogliere le uova (e carcasse) per centrifugazione a 450 xg per 5 min. </li><li> Lavare le uova con tampone sterile uovo 2-3x. </li><li> Incubare le uova di O / N in tampone M9 e loro su piastre NGM standard di seme. </li></ol></li><li> Saggio di chemiotassi. <ol><li> Preparare diversi pezzi di agar circa 0.5x0.5x0.5 cm. Abbiamo depositare l&#39;agar-agar in un piatto da 10 cm e abbiamo tagliato i pezzi di agar da lì. </li><li> Mettere a bagno il pezzo agar in una soluzione contenente l&#39;attrattivo desiderata (a saturazione, le concentrazioni di quasi saturazione) per 2 ore. Attrattivi tipici sono lisina (0,5 M), biotina (0,2 M). </li><li> Depositare un chunk agar in una piastra di prova 10 cm, in cui la posizione di un luogo di prova e un punto di controllo sono stati contrassegnati (Figura 1A). Raggiungere l&#39;equilibrio e la formazione di un gradiente di O / N (Figura 1B). Preparare 5 piastre per un singolo esperimento. </li><li> Prima dell&#39;esperimento aggiungere 10 ml di 20 mM NaN 3 (Anesthetic) per ogni spot. </li><li> Collocare 20 vermi età sincronizzate nel centro della piastra. Porre la piastra in incubatore a 20 ° C. </li><li> Dopo 1 ora, contare gli animali sui punti di prova / controllo e di calcolare l&#39;indice di chemiotassi, (CI) come segue: <img alt="Equazione 1" fo:content-width=".863in" fo:src="/files/ftp_upload/50435/50435eq1highres.jpg" src="/files/ftp_upload/50435/50435eq1.jpg" /> dove N, N e N Cnt prova., indicano il numero totale di animali, il numero di animali nel punto di prova e il numero di animali nel punto di controllo. </li></ol></li></ol> 3. Primaria Embryonic Cell Culture <ol><li> Lyse vermi come descritto in età-sincronizzazione. </li><li> Arrestare la reazione di lisi aggiungendo lo stesso volume di tampone sterile uovo e centrifugare a 450 xg per 5 min. Scartare il surnatante delicatamente facendo attenzione a non perdere le uova pellet. Ripeti 2-3x o fino a quando il surnatante è chiaro. </li><li> Risospendere pellet (uova e carcasse) In 2 ml di tampone sterile uovo e aggiungere 2 ml di soluzione sterile di 60% di saccarosio in tampone uovo. Mescolare questa soluzione fino a quando le uova sono completamente risospese (in quanto tendono a formare grumi sotto la centrifugazione) a mano o con il vortex. </li><li> Centrifugare a 450 xg per 15 min. </li><li> Trasferire accuratamente surnatante (contenente le uova) in una provetta sterile. Scartare pellet che contiene le carcasse e di altri sottoprodotti di lisi. </li><li> Rimuovere saccarosio residuo risospendendo le uova in tampone uovo e centrifugazione a 450 xg per 5 min. Raccogliere delicatamente e scartare il surnatante. Ripetere 3 volte. </li><li> Sotto una cappa laminare sospendere di nuovo le uova pellet in tampone uovo sterile contenente 1 U / ml chitinasi a RT per digerire i gusci d&#39;uovo. Dopo 30 min iniziare a monitorare la reazione (sotto un microscopio coltura cellulare invertito). Ogni serie di chitinasi ha un&#39;attività leggermente diversa. Tipicamente digestione è completato in 1 ora. </li><li> Quando circa 70-80% gusci d&#39;uovo vengono digeriti da chitinasi add CM-15 (L-15 di coltura cellulare medium contenente 10% siero fetale bovino, 50 U / ml di penicillina, e 50 ug / ml streptomicina). Dissociare le cellule usando una siringa con una calibro 27. Filtrare la sospensione cellulare con un 5,0 micron filtro per eliminare gli embrioni intatti, grumi di cellule e larve. </li><li> Pellet la sospensione cellulare dissociato mediante centrifugazione a 450 xg per 15 min. Rimuovere il surnatante e risospendere il pellet in CM-15 terreno di coltura cellulare. </li><li> Piastra cellule dissociate sul coperchio di vetro scivola precedentemente rivestiti con arachidi lectina (0,1 mg / ml) disciolto in acqua. Nota: le cellule devono aderire al substrato al fine di differenziare. </li><li> Cellule possono essere mantenute a TA (16-20 ° C) in aria per più di 2 settimane. </li></ol>

<ol>
	<li>Hardy, J., Allsop, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposition+as+the+central+event+in+the+aetiology+of+Alzheimer's+disease.">Amyloid deposition as the central event in the aetiology of Alzheimer's disease.</a> Trends in Pharmacological Sciences. 12 (10), 383-388 (1991).</li><li>Kotzbauer, P. T., Trojanowsk, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+pathology+in+Alzheimer's+disease.">Lewy body pathology in Alzheimer's disease.</a> Journal of Molecular Neuroscience. 17 (2), 225-232 (2001).</li><li>Greenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+myositis:+review+of+recent+literature.">Inclusion body myositis: review of recent literature.</a> Current Neurology and Neuroscience Reports. 9 (1), 83-89 (2009).</li><li>Revesz, 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=Sporadic+and+familial+cerebral+amyloid+angiopathies.">Sporadic and familial cerebral amyloid angiopathies.</a> Brain Pathology. 12 (3), 343-357 (2002).</li><li>Hartmann, 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=Distinct+sites+of+intracellular+production+for+Alzheimer's+disease+A+beta40/42+amyloid+peptides.">Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.</a> Nature Medicine. 3 (9), 1016-1020 (1997).</li><li>Ohnishi, S., Takano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+fibrils+from+the+viewpoint+of+protein+folding.">Amyloid fibrils from the viewpoint of protein folding.</a> Cellular and Molecular Life sciences: CMLS. 61 (5), 511-524 (2004).</li><li>DL, R. i. d. d. l. e., 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=C.+elegans+II.">C. elegans II.</a> Cold Spring Harbor Monograph. 1, 1222 (1997).</li><li>Cotella, 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=Toxic+role+of+k++channel+oxidation+in+Mammalian+brain.">Toxic role of k+ channel oxidation in Mammalian brain.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 32 (12), 4133-4144 (2012).</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> Proceedings of the National Academy of Sciences of the United States of America. 92 (20), 9368-9372 (1995).</li><li>Cai, S. Q., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+a+potassium+channel+causes+progressive+sensory+function+loss+during+aging.">Oxidation of a potassium channel causes progressive sensory function loss during aging.</a> Nature Neuroscience. 12 (5), 611-617 (2009).</li><li>Yu, S., 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=Guanylyl+cyclase+expression+in+specific+sensory+neurons:+a+new+family+of+chemosensory+receptors.">Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 3384-3387 (1997).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7 (5), 729-742 (1991).</li><li>Caserta, T. 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=Q-VD-OPh,+a+broad+spectrum+caspase+inhibitor+with+potent+antiapoptotic+properties.">Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties.</a> Apoptosis : an international journal on programmed cell death. 8 (4), 345-352 (2003).</li><li>Christensen, 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=A+primary+culture+system+for+functional+analysis+of+C.+elegans+neurons+and+muscle+cells.">A primary culture system for functional analysis of C. elegans neurons and muscle cells.</a> Neuron. 33 (4), 503-514 (2002).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui si descrive un approccio combinato, per studiare gli aspetti cellulari e molecolari delle amylopathies utilizzando C. elegans. I vantaggi di questo approccio includono:. 1) basso costo C.elegans è mantenuta in normali Petri seminato con batteri, a temperatura ambiente. 2) genetica potente. Animali transgenici possono essere ottenuti in pochi mesi e alla vasta gamma di sequenze promotore è disponibile per guidare l&#39;espressione del gene desiderato in neuroni specifici. 3), ben caratterizzato, s...

Beta amiloide (Ap) è un peptide di 36-43 aminoacidi che si forma a seguito della scissione sequenziale della proteina precursore dell&#39;amiloide (APP) per β e γ secretasi 1. Il γ secretasi elabora l&#39;estremità C-terminale del peptide Ap ed è responsabile per le sue lunghezze variabili 5. Le forme più comuni di Ap sono Ap 40 Ap e 42, quest&#39;ultimo essendo più comunemente associato a condizioni patologiche come 5 AD. Ad alte concentrazioni Ap forma β-sheets che aggregano a formare fibrille amiloidi 6. Depositi di fibrille sono il componente principale delle placche senili che circondano i neuroni. Entrambe le targhe e diffusibili, oligomeri Ap non placca, si pensa di costituire le forme patogene sottostanti Ap. Studio di laboratorio di amylopathies neuronali è complicata dal fatto che queste condizioni progresso con il tempo. Pertanto, è important di sviluppare modelli animali geneticamente trattabili-complementari a topi-con vita breve. Questi modelli possono essere utilizzati per spiegare aspetti specifici di amylopathies-tipicamente cellulare e molecolare e in virtù della loro semplicità, aiutare a catturare l&#39;essenza del problema. Il verme Caenorhabditis elegans cascate è questa categoria. Ha una durata di vita breve, ~ 20 giorni e in aggiunta i processi cellulari di base tra cui regolazione dell&#39;espressione genica, traffico di proteine, connettività neuronale, sinaptogenesi, segnalazione cellulare e la morte sono simili ai mammiferi 7. Le caratteristiche uniche del worm includono la genetica potenti e la mancanza di un sistema di vasi, che permette di studiare i danni neuronali indipendentemente da danno vascolare. D&#39;altra parte, la mancanza di un cervello limita l&#39;uso di C. elegans per studiare molti aspetti della neurodegenerazione. Inoltre, la riproduzione e identificazione di distribuzioni anatomiche delle lesioni non possono essere eseguite in questo organismo. Altro limitezioni comprendono la difficoltà di valutare sia le differenze nei profili di espressione genica e di valore di un comportamento complesso e funzione di memoria. Qui si descrivono i metodi per generare C. elegans modelli di amylopathies.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1. NGM</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td>17 g</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>AMRESCO</td>
			<td>J636</td>
			<td>2.5 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 975 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving, then add the following items and mix well</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C3045</td>
			<td>1 ml of 5 mg/ml stock( in ethanol)</td>
		</tr>
		<tr>
			<td>Potassium phosphate buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>25 ml of 1M stock</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Potassium phosphate buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>108.3 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>35.6 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. Egg buffer (pH 7.3, 340 mOsm)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>118 mM</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>48 mM</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M4880</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. CM-15</td>
		</tr>
		<tr>
			<td>L-15 culture medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>450 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 units/ml</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 g/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. Other Reagents</td>
		</tr>
		<tr>
			<td>Halocarbon 700 oil</td>
			<td>Halocarbon Products</td>
			<td>9002-83-9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 &mu;m Acrodisc Syringe Filter</td>
			<td>PALL Co.</td>
			<td>4199</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-5UN</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lectin (peanut)</td>
			<td>Sigma-Aldrich</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
			<td>425044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>71289</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a caenorhabditis elegans model system for amylopathy study

Amylopathy è un termine che descrive la sintesi e l&#39;accumulo anomalo di beta amiloide (Ap) nei tessuti con tempo. Ap è un segno distintivo della malattia di Alzheimer (AD) e si trova nella demenza a corpi di Lewy, miosite da corpi inclusi e angiopatia amiloide cerebrale 1-4. Amylopathies progressivamente si sviluppano con il tempo. Per questo motivo gli organismi semplici con brevi durate di vita possono aiutare a chiarire gli aspetti molecolari di queste condizioni. Qui, descriviamo protocolli sperimentali per lo studio neurodegenerazione Ap-mediata utilizzando verme Caenorhabditis elegans. Così, costruiamo vermi transgenici iniettando il DNA codificante Ap umano 42 nelle gonadi sinciziale di ermafroditi adulti. Linee trasformanti sono stabilizzate da una integrazione mutagenesi indotta. Nematodi sono sincronizzati età raccogliendo e seminando loro uova. La funzione dei neuroni che esprimono Ap 42 viene testato in saggi comportamentali opportune (chemiotassi assays). Colture primarie neuronali ottenute da embrioni vengono utilizzati per integrare i dati comportamentali e per testare gli effetti neuroprotettivi di composti anti-apoptotici.

Con i nostri protocolli si studiano gli effetti di Ap umano 42 oligomero sulla funzione neuronale 8. Un frammento codificante Ap umano 42 e il peptide segnale codificante sequenza artificiale di fuoco vettore pPD50.52 è stato amplificato dal costrutto PCL12 9 utilizzando primer che hanno introdotto un sito di endonucleasi di restrizione 1 Sma alle estremità. Il frammento è stato poi inserito in un costrutto contenente un FLP-6 sequenza promotrice 2.481 bp nel fuoco...

Watch this Scientific Journal Video about A Caenorhabditis elegans Model System for Amylopathy Study at JoVE.com

A Caenorhabditis elegans Model System for Amylopathy Study

Descriviamo i metodi per studiare aspetti della amylopathies nel verme<em&gt; C. elegans</em&gt;. Mostriamo come costruire i vermi che esprimono Ap umano<sub&gt; 42</sub&gt; Nei neuroni e come testare la loro funzione in saggi comportamentali. Mostriamo ulteriormente come ottenere colture neuronali primarie che possono essere utilizzati per il test farmacologico.

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 ...

a-caenorhabditis-elegans-model-system-for-amylopathy-study

Research

JoVE Journal

Neuroscience

A Caenorhabditis elegans Model System for Amylopathy Study

10.4K Views.  University of Medicine and Dentistry of New Jersey. Descriviamo i metodi per studiare aspetti della amylopathies nel verme C. elegans. Mostriamo come costruire i vermi che esprimono Ap umano 42 Nei neuroni e come testare la loro funzione in saggi comportamentali. Mostriamo ulteriormente come ottenere colture neuronali primarie che possono essere utilizzati per il test farmacologico.

Video: A Caenorhabditis elegans Model System for Amylopathy Study

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

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

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

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

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 ...

Ricerca

Neuroscienze

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Metodi per studiare i meccanismi d&#39;azione di farmaci antipsicotici in<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Il sistema olfattivo come modello per studiare gli schemi di crescita assonale e Morfologia<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un test per misurare gli effetti di etanolo sulla velocità di Locomotion<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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 ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Nuovi parametri per caratterizzare embrionale Allungamento del nematode<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UN<em&gt; Caenorhabditis elegans</em&gt; Analisi di Aversione di rame basata sullo stato nutrizionale

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Raziometrici calcio Imaging di singoli neuroni nel comportarsi Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Imaging delle spine dendritiche in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

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

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

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

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

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

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