Questi studi sono stati supportati da sovvenzioni dal National Institutes of Health, Istituto Nazionale per l&#39;alcolismo e abuso di alcool: R01AA016837 (JCB) e P20AA017828 (AGD e JCB).

1. procedura per eseguire il giorno prima del test <ol><li> Scegli L4 vermi stadio a fresco mezzo di crescita nematode (NGM) piastre seminate con un prato di OP50 E. coli, e la cultura a 20 CO / N. Ogni condizione test richiede 10 vermi; scegliere un eccesso di vermi per consentire O / N perdita di vermi. <ol><li> Animali solo test che sono gli adulti del primo giorno; molti mutanti crescono a una velocità più lenta di tipo selvaggio. Regolare i tempi di raccolta per i ceppi che hanno ritardi di sviluppo in modo che tutti gli animali testati sono adulti primo giorno. </li></ol></li></ol> 2. procedura per eseguire il Giorno del saggio <ol><li> Preparazione per il saggio: <ol><li> Effettuare test su 60 x 15 millimetri piastre non teste di serie standard di NGM. Asciugare tutti piatti da utilizzare a 37 ° C per 2 ore, con coperchio off. Per ogni prova sperimentale, utilizzare quattro piastre NGM secchi; questi saranno gli 0 e 400 mm piastre test etanolo e i loro piatti di acclimatazione di accompagnamento. NOTE:L&#39;uso di piastre NGM è importante; differenze nella composizione delle piastre, in particolare la loro osmolarità, possono influenzare fortemente la risposta dose di etanolo sul comportamento, ciò è dovuto, almeno in parte, ai cambiamenti nella quantità di etanolo accumulata dagli animali 20. NGM agar è di 160 mOsm. </li><li> Dopo l&#39;essiccazione, pesano ciascuna delle piastre NGM non teste di serie da utilizzare nel dosaggio e nota il peso. Determinare il volume del supporto nelle piastre base al peso di un piatto vuoto. Per approssimare la conversione del peso del agar a volume, si supponga che il supporto pesa la stessa di un uguale volume di acqua. NOTA: I nostri risultati più consistenti sono stati trovati con i media NGM che ha disidratati sufficientemente che un volume originale 10 ml ha un volume di post-essiccazione tra 8,3-8,9 ml. Un&#39;alternativa al tempo secco 2 hr è asciugare le piastre fino a raggiungere questa gamma volume per tenere conto delle differenze in incubatrici. </li><li> Sciogliere 4 anelli di rame (diametro interno di 1.6 cm) nella superficie di ciascuna delle piastre di agire come recinti per i diversi genotipi o gruppi di trattamento di worms.Grasp ogni anello con una pinza, e calore in una fiamma (forte fiamma di un becco Bunsen funziona bene) per circa 3 sec. Porre immediatamente l&#39;anello su un piatto, mentre ancora in mano l&#39;anello con le pinze per evitare che &#39;saltare&#39;. <ol><li> Assicurarsi che l&#39;anello poggia piatta contro la superficie del agar premendo leggermente con la pinza in diversi punti sull&#39;anello. Quando si posiziona l&#39;anello, attenzione a lasciare spazio per altri tre anelli. </li><li> Sciogliere i tre anelli di rame aggiuntivi nella superficie della piastra, avendo cura di posizionare il più vicino possibile tra loro in modo che tutti e quattro sarà nel campo visivo della telecamera. NOTA: Fare una buona tenuta con l&#39;agar è essenziale per mantenere i vermi negli anelli durante il dosaggio. Anelli che saltano intorno sul piatto è improbabile che rendere la tenuta corretta con l&#39;agar epuò cicatrice l&#39;agar, che permette vermi scavano e interferisce con la visualizzazione dei vermi. </li><li> Per ogni piatto saggio preparare un piatto di accompagnamento &quot;acclimatazione&quot;, che dovrebbe essere essiccata e testa di serie e riceverà nessun etanolo. Mettere quattro anelli di rame su queste tavole. </li></ol></li><li> Etichettare il fondo delle piastre adiacenti anello con il ceppo vite senza fine per essere utilizzato nell&#39;anello, avendo cura di non scrivere nel campo visivo dell&#39;anello stesso. Far corrispondere le etichette sulla piastra test con la piastra di acclimatazione di accompagnamento. </li><li> Calcolare la quantità di etanolo al 100% da aggiungere a ciascuna piastra di test in modo che la concentrazione finale è 0 mM o etanolo mM 400 (peso a volume). Per ogni esperimento (n = 1), ci sarà un 0 mM ed una piastra 400 mM etanolo; piastre acclimatazione non ricevono etanolo. Aggiungere l&#39;etanolo, pipettando intorno alla superficie della piastra. Utilizzare pellicola di laboratorio per sigillare la piastra e lasciarlo equilibrare sul banco per 2 ore. </li><li> Trascorso 1,5 ore, iniziare la fase di acclimatazione del dosaggio, passo 2.2. </li></ol></li><li> Eseguire test locomozione: lastre Assay <ol><li> Trasferire accuratamente 10 vermi di ogni gruppo sperimentale al centro di un anello di rame sulla piastra acclimatazione. Rimuovere qualsiasi alimento che è visibile sull&#39;agar a questo punto raschiando delicatamente con il pick verme. Variare l&#39;ordine in cui i gruppi sperimentali sono messi sulle piastre attraverso prove sperimentali in modo che gli stessi ceppi non vengono messi sulle piastre nello stesso ordine in tutti gli studi. NOTA: L&#39;obiettivo è quello di trasferire gli animali alla piastra con quantità minime di cibo, perché se cibo sul piatto acclimatazione viene trasferito alla piastra di dosaggio i vermi aggregare attorno al cibo e l&#39;effetto del farmaco sulla locomozione sarà oscurata. </li><li> Fare attenzione a non rompere la superficie del agar con il plettro, in quanto questo permetterà ai vermi di tana e interromperà il dosaggio. Incubare i vermi a temperatura ambiente per 30 min. </li><li> Assicurarsi che vi sia un intervallo adeguato tra le iniziazioni di ogni piatto. Uno sperimentatore esperto può muovere 40 animali, 10 / anello per 4 anelli in &lt;1,5 min, ma qualunque intervallo fino a 2 minuti è accettabile. Il test standard è la registrazione di filmati a 10-12 min e 30-32 minuti di esposizione sia per 0 mm e 400 mm di etanolo. </li><li> Avviare la piastra acclimatazione per l&#39;esposizione 0 mm e la piastra di acclimatazione per l&#39;esposizione 400 mm circa 2 min e 30 sec a parte per consentire il risparmio del primo filmato prima del secondo filmato deve iniziare. </li><li> Dopo il periodo di acclimatazione di 30 min, trasferire i vermi dalle piastre acclimatazione alle piastre di saggio. Trasferire i vermi alle piastre di saggio (0 mM o etanolo 400 mM) nello stesso ordine in cui sono stati aggiunti alla piastra acclimatazione, tenere traccia del tempo tra i completamenti di ogni piastra. Sigillare la piastra con la pellicola di laboratorio per minimizzare la perdita di etanolo per evaporazione. </li><li> USE un movimento scavare con un pick verme sottile taglio appiattito per raccogliere i vermi in cima alla scelta appiattito. Eseguire il trasferimento di vermi dalla piastra acclimatazione unseeded alla piastra dosaggio senza l&#39;uso di batteri, che è comunemente usato nel trasferimento vermi quanto contribuisce a incollare il verme al ritiro. NOTA: La velocità alla quale gli animali vengono trasferiti in questo passaggio è molto importante perché le prime vermi sulla piastra saranno esposti ad etanolo più lungo degli ultimi vermi aggiunti alla piastra, e precedenti punti di tempo può mostrare alcuni effetti dipendenti dal tempo. Questa è la ragione principale per la quale i ceppi rotazione sono posti su una piastra prima attraverso repliche sperimentali. </li></ol></li><li> Eseguire Locomotion Assay: riprese <ol><li> Utilizzare una combinazione microscopio / macchina che permette l&#39;imaging simultaneo di tutti i quattro anelli nel campo visivo (42x42 mm 2 circa quadrata), ad esempio un obiettivo microscopio 0.5x, 0.8x ingrandimento e una fotocamera con 2048x2048 7.481; m CCD pixel. </li><li> Utilizzare illuminazione uniforme in tutto il campo di vista, che aiuta a riconoscimento di oggetti nella fase soglia di intensità (vedi sotto). A 3 &quot;x3&quot; retroilluminazione funziona bene. </li><li> Immagine i vermi su una piastra di supporto posizionata rivolta verso l&#39;alto (coperchio verso il basso), che genera contrasto che si perde quando si usa la retroilluminazione rispetto ad un microscopio tradizionale trasmessa sorgente luminosa. </li><li> Preparare il software di analisi di immagine per catturare filmati time-lapse dei vermi in movimento. Impostare il software per catturare le immagini in scala di grigi a 12 bit ogni 1 sec, come 2-min (120 frame) del filmato. Per ridurre la dimensione del file di output, pur mantenendo la risoluzione dell&#39;immagine sufficiente, utilizzare una modalità binning 2x2 per catturare immagini 1024x1024 pixel. </li><li> Registrare i film. Iniziare la registrazione di un filmato 120-frame della prima piastra (0 etanolo mM) 10 min dopo gli ultimi vermi sono stati posti su quel piatto. Salvare il file del filmato. </li><li> Registrare il secondo piatto (etanolo 400 mm). Ripetere questo processo perentrambi i piatti iniziano 30 minuti dopo le ultime vermi sono stati collocati sul primo piatto (0 etanolo mm) per catturare i 30-32 punti min di tempo per ogni esposizione. NOTA: Lasciare il tempo sufficiente per salvare i file di immagine dopo ogni sessione di acquisizione prima di iniziare la registrazione della targa accanto da analizzare. </li><li> Per il futuro-proofing di film archiviati e permettere questi film di essere aperti e analizzati da altri programmi software di imaging open source di dominio pubblico, come ImageJ 28, convertire una copia di ogni film a 8-bit 256 file TIFF in scala di grigi. NOTA: Il software di analisi delle immagini qui descritto utilizza un formato di file proprietario. </li></ol></li><li> Analisi di filmati utilizzando software ImagePro. <ol><li> Una volta catturato, analizzare i filmati utilizzando Immagine-Pro Plus software o il suo equivalente. NOTA: Una varietà di altri software di monitoraggio oggetto è disponibile che è stato utilizzato per monitorare con successo molteplici C. elegans contemporaneamente 29 e talesoftware potrebbe ragionevolmente sostituire quello descritto qui come le misure di identificazione degli oggetti si basano su principi simili. Il metodo descritto si riferisce alle versioni Immagine-Pro Plus software 6,0-6,3 e 7.0, le versioni più recenti di Immagine-Pro Plus software può avere piccole differenze. </li><li> Analizzare i film in 2-min (120 frame) segmenti. Innanzitutto, applicare un filtro alle immagini per appiattire lo sfondo e migliorare il contrasto degli oggetti vermi. Selezionare il filtro come segue: Menu&gt; Processo&gt; Filtri&gt; Miglioramento&gt; appiattire: Parametri: background = scuro; Caratteristica Width = 20 Pix. </li><li> Re-salvare i filmati dopo la fase di filtraggio, ma mantenere sempre il film originale nella sua forma non filtrato. <ol><li> Analizzare la locomozione di animali in ciascun anello separatamente con una regione circolare di interesse (ROI) che è posto e dimensionata per sovrapporsi con l&#39;anello di rame. Identificare e seguire i vermi con il Menu&gt; Misura&gt; Traccia oggetti ... di comando; questo porta il monitoraggio Dfinestra Tabella ata. Il pulsante Opzioni di monitoraggio permette brani specifici da escludere e per limitare eventuali artefatti sperimentali. </li></ol></li><li> Nella scheda Auto Tracking, utilizzare i seguenti parametri: parametri di traccia: il limite di velocità (il raggio di ricerca) = 400 micron / telaio, limite di accelerazione = Auto, Lunghezza minima traccia totale = 400 micron, predominante tipo di movimento = caotico; Oggetti in parametri tracce: Lasciare tracce parziali = yes, lunghezza minima = 21 fotogrammi, Carrellata profondità previsione = 1 fotogramma. </li><li> Per avviare il processo di monitoraggio, fare clic sul Trova tutte le canzoni automaticamente tasto di funzione per aprire la finestra di dialogo Count / opzioni di formato e la finestra di dialogo di monitoraggio. Selezionare l&#39;opzione manuale per la Selezione campo di intensità nella finestra di dialogo Conte / dimensione, che fornisce l&#39;importante passo soglia che utilizza l&#39;intensità in scala di grigi del worm pixel per evidenziare un oggetto verme di analisi e di ignorare i precedenti più leggeri pixel. <ol><li> Regolare la sli soglia intensitàders (uno per impostare il limite superiore, uno per impostare il limite inferiore) per creare un intervallo compreso che mette in evidenza tutti gli oggetti scuri. Una gamma di 0-1,500 su una scala da 0-4,095 è un buon punto di partenza per la sintonizzazione fine. </li><li> Applicare un filtro per escludere dimensioni oggetti che sono più grandi e più piccolo di un singolo oggetto senza fine, come l&#39;anello di rame e detriti sulle piastre. Impostare due parametri di dimensione per fare questo che riguardano le dimensioni dei singoli vermi in una varietà di posizioni con la misura&gt; Select Misure voce di menu nella finestra di dialogo Opzioni Count / Taglia. (Range Area: 28,000-120,000 micron 2 e Fascia perimetrale: 600-2,500 micron). </li><li> Se un ceppo mutante è significativamente più piccolo o più grande di altri animali sulla piastra da analizzare, ampliare la gamma delle impostazioni di filtro per il riconoscimento di oggetti per accogliere le diverse dimensioni dei ceppi prima di inseguimento animali così che le impostazioni non devono essere modificate mid-analisi. </li></ol> &lt;/ Li&gt; <li> Completare il processo di monitoraggio facendo clic su Continua nella finestra di dialogo di monitoraggio. Confrontare visivamente le piste di uscita con il progresso di ogni verme nel film per assicurare che ogni worm è rappresentato meno che ci sia un motivo valido per escludere che in base alle impostazioni del filtro automatico. Eliminare manualmente brani che sono stati prodotti dalla presenza di oggetti confermati non vermi che hanno soddisfatto i criteri di filtro dimensioni. </li><li> Utilizzare il software per calcolare la velocità di ogni verme tra ogni fotogramma (distanza percorsa per il baricentro di un oggetto per 1 sec tra i fotogrammi) e visualizza la velocità media per ogni traccia e la velocità media di tutte le tracce per la popolazione di vermi in anello di rame. Considerate questo media finale sia n = 1 in termini di prove sperimentali. Esportare i dati in un foglio di calcolo per analisi statistiche e l&#39;archiviazione dei dati. </li><li> Una volta che i dati sono stati registrati per il primo anello sulla piastra, spostare la ROI all&#39;anello successivo eripetere il processo di monitoraggio, senza modificare alcun parametro. </li><li> Calcola la velocità relativa di locomozione attraverso: </li><li> Velocità relativa (%) = trattati (400 mm) Velocità / grezzo (0 mm) Velocità x 100. NOTA: diverse manipolazioni genetiche spesso alterano il tasso di locomozione basale di animali. Al fine di determinare l&#39;effetto di etanolo sulla velocità locomozione degli animali e per poter confrontare questi effetti attraverso diversi genotipi o condizioni, calcolare una velocità relativa. </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., 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=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

La semplice neurobiologia e strumenti genetici disponibili in C. elegans rendere il worm un ottimo modello in cui studiare le basi molecolari degli effetti di etanolo sul comportamento. Qui, descriviamo un metodo che è stato utilizzato per individuare alcuni mediatori molecolari ed ambientali della risposta comportamentale acuta etanolo 8,10,20,24,25. Questo metodo consente la differenziazione e l&#39;esame simultaneo di due diversi fenotipi comportamento di risposta etanolo, sensibilità iniziale e...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

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.

Dati rappresentativi (Figura 1) da diversi genotipi differenti ed i loro controlli appaiati vengono presentati 8,24; i dati sono stati appositamente scelti che le differenze evidenziano negli animali testati. Il grado di effetto a 10 minuti di esposizione è considerata la sensibilità iniziale di un ceppo, che è visualizzata sullo assi sinistra in Figura 1B-G. Ceppi mutanti con una velocità relativa più grande del controllo a 10 min sono considerati resistenti (Fi...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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 ...

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Neuroscience

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

8.5K Views.  Virginia Commonwealth University. 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. 

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

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialisi di etanolo durante la determinazione operante auto-amministrazione ed etanolo etanolo mediante gascromatografia

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Ricerca

Neuroscienze

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Un saggio per permeabilità del neuroepitelio Zebrafish embrionali

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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 ...

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.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Un poco costoso, test comportamentale scalabile per la misurazione etanolo sedazione sensibilità e tolleranza Rapid in<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

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. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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 ...