We thank the Duke Physics Shop, especially Phil Lewis, for helping us build the behavioral apparatus and attachments and creating the drawing. This work is funded by the National Institutes of Health under award number R01GM100027.

1. Preparazione mosche da dosare <ol><li> Cultura vola di melasso normali / media farina di mais in incubatore a 25 ° C e 65% di umidità. Fare attenzione a non affollare i flaconi. Per esempio, mettere 8 femmine e 6 maschi in una stretta fiala di cibo. Nota: Il &quot;flaconcino cibo stretta&quot; qui utilizzato ha un diametro interno di 2,3 cm. Noi di solito dispensati circa 10 ml di cibo volare in ogni fiala. La ricetta cibo mosca che abbiamo usato è descritto qui: http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html. </li><li> 2 - 3 giorni dopo femmine eclosion, preparare fiale con pasta di lievito fresca come mostrato nella Figura 1E. Rendere pasta di lievito fresca miscelando 6 g di lievito attivo con 10 ml di acido proprionic 0,5%. Utilizzare una spatola per applicare la pasta di lievito sul fianco delle fiale alimentari. Raccogliere 30 - 35 femmine eclosed insieme a 20 - 25 maschi nelle fiale. <ol><li> In generale, raccogliere i maschi dalle stesse fiale in cui sono raccolti le femmine(In modo da risparmiare tempo e fatica). Tuttavia, se non vi è preoccupazione per la fertilità dei maschi, utilizzare i maschi WT. Inoltre, pasta di lievito è importante per stimolare la produzione di uova quindi prepararlo fresco ogni giorno. </li></ol></li><li> Mantenere la raccolta femmine / maschi a 25 ° C, 65% di umidità, a meno che le mosche sono sensibili alla temperatura. </li><li> Dopo ~ 4 - 5 giorni, controllare le fiale per vedere se le femmine sono pronti per esperimenti di deposizione delle uova. Sono pronti quando la superficie del mezzo cibo è bagnato dalla larve scavando attivamente il cibo (vedi Figura 1E - F). Nota: Le femmine di solito trattengono uova, che una volta che la superficie del cibo diventa bagnata e brulicante di larve. Questo passaggio garantisce che le femmine sarebbero pronti a deporre molte uova quando incontrano substrati con consistenza desiderata (ad es., ~ 1% agarosio). </li></ol> 2. Camera di costruzione, di montaggio, di installazione Assay <ol><li> Avere un negozio di macchina costruire camere di deposizione delle uova in acrilico (Fifigura 1A - D). Il disegno di ingegneria di diversi pezzi sono stati mostrati in figura supplementare 1 -. 3 immagini ad alta risoluzione si possono trovare anche qui (http://www.rebeccayang.org/pdf/chamber%20design.pdf). </li><li> Inserire fogli di plastica in carico (alto) pezzo della camera, come mostrato nella Figura 1C. Questo serve come la superficie inferiore durante il caricamento vola in singole arene deposizione delle uova. </li><li> Anestetizzare le femmine su un rilievo di CO 2 e caricarli singolarmente in ogni arena deposizione delle uova. Consentire ~ 30 min per le mosche di recuperare dal CO 2 e per acclimatarsi al nuovo ambiente. </li><li> Preparare i substrati agarosio. <ol><li> Per comodità, tenere una bottiglia premade del fuso 1% agarosio a bagnomaria C 55 °. </li><li> Aggiungere la quantità desiderata di soluzione madre di saccarosio (2 M) in una provetta conica da 50 ml e mescolandolo con quantità appropriata di agarosio. Ad esempio, per preparare il substrato 150 mM saccarosio, place 750 ml di 2 M soluzione di saccarosio nel tubo e quindi riempire il tubo con agarosio al segno 10 ml. </li><li> Preparare il substrato pianura nello stesso modo ma aggiungere acqua distillata al posto della soluzione di saccarosio. Nota: La concentrazione finale di agarosio in questo protocollo è leggermente inferiore a 1%. Nella nostra esperienza, l&#39;esatta concentrazione di agarosio non importa fintanto che viene controllato per essere all&#39;interno di ~ 0,9 - 1,1% e che i due substrati sono della stessa concentrazione di agarosio. </li></ol></li><li> Prendere il pezzo della camera e pipetta 1000 ml di substrato agarosio substrato (in basso) in ciascun canale, come mostrato nella Figura 1D. </li><li> Lasciare agarosio solidificare per ~ 30 min. </li><li> Una volta che i substrati di agarosio e mosche sono pronti, assemblare tutti i tre pezzi della camera di deposizione delle uova e poi prendere i fogli di plastica. </li><li> Mettere le camere in incubatrici mosca. Nota: Lunghezza di esperimenti di deposizione delle uova può variare a seconda espeesigenze rimental. Noi di solito Simula O / N (14 - 16 ore). Inoltre, è stato osservato alcun impatto significativo dei tempi circadiano sulle preferenze di deposizione delle uova. </li><li> Anestetizzare le femmine iniettando CO 2 nella camera. Smontare la camera, scartare le mosche anestetizzati in volo all&#39;obitorio (es., Un caffè vuota può riempito con alcuni olio di mais). Prendere le immagini dei risultati per registri (vedi figura 2). </li><li> Contare il numero di uova manualmente e calcolare gli indici di preferenza per l&#39;analisi. Calcolare l&#39;indice preferenza come (N a - N b) / (N + N un b) dove N e N un B rappresentano il numero di uova in loco un sito vs. b, rispettivamente. </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg-laying+demand+induces+aversion+of+UV+light+in+Drosophila+females.">Egg-laying demand induces aversion of UV light in Drosophila females.</a> Curr. Biol. 24 (23), (2014).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat. Methods. 6, 451-457 (2009).</li><li>Klatpoetke, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Optical+Excitation+of+Distinct+Neural+Populations.">Independent Optical Excitation of Distinct Neural Populations.</a> Nat. 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The authors have nothing to disclose.

Le camere e protocolli descritti qui sono diversi miglioramenti rispetto ai precedenti test di deposizione delle uova. Innanzitutto, aumentano la velocità di saggiare preferenze dei singoli animali significativamente. Ogni camera può saggiare 30 singole femmine e richiede meno di un&#39;ora di set up. In secondo luogo, aumentano la consistenza delle preferenze di deposizione delle uova rispetto ai metodi precedenti. La standardizzazione delle dimensioni dell&#39;arena, le dimensioni dei substrati deposizione delle uova, e la distanza tra i substrati rende più facile per confrontare i risultati tra i diversi giorni sperimentali e da diversi gruppi di ricerca. Terzo, le camere possono essere dotate di attacchi per accogliere ulteriori analisi si vuole perseguire (figura complementare 4). Ad esempio, per l&#39;analisi comportamentale, telecamere possono essere collegati alla sommità della camera di registrare le mosche 7,11-13. Ancora più importante, questo approccio è scalabile. Queste camere sono relativamente inexpensieroso per produrre con l&#39;aiuto di una officina meccanica. Inoltre, si richiede in genere un operaio specializzato a meno di 2 ore per impostare 5 camere (150 singole femmine) vale la pena di saggi di deposizione delle uova. Uno dei fattori più critici quando si studia la preferenza deposizione delle uova delle singole femmine è quello di garantire che ogni femmina è innescato a deporre molte uova. (Una preferenza di &quot;50 contro 0 uovo&quot; è molto più convincente di una preferenza di &quot;1 vs 0 uovo&quot;). Questo protocollo, se eseguito correttamente, dovrebbe consentire mosche wild-type regolari (Canton S e w1118) determinare almeno 50 uova / per femmina O / N. Diversi fattori contribuiscono alla produzione di femmine con alto tasso di deposizione delle uova. In primo luogo, si dovrebbe fare attenzione a non sovrappopolare le fiale / flaconi quando la coltura delle femmine da dosare. Il sovraffollamento produce spesso larve più piccoli, che crescono in femmine più piccole che depongono meno uova. In secondo luogo, si dovrebbe fare in modo che le femmine di essere analizzati hanno accesso a un sacco di pasta di lievito quando sono mescolati insieme ingegnoh maschi in fiale alimentari. Si raccomanda che l&#39;acido proprionic 0,5% al ​​posto dell&#39;acqua da utilizzare per preparare pasta di lievito perché l&#39;acido proprionic aumenta non solo ovodeposizione ma riduce anche infezione fungina in fiale. In terzo luogo, non si deve saggiare le femmine fino a quando la superficie di larve / cibo del cibo fiale guardare come l&#39;immagine mostrata in figura 1F; se non sufficientemente deposizione delle uova privato, le femmine non avrebbero deporre il maggior numero di uova quando dosati nelle camere. Si noti inoltre che le fiale alimentari usati qui sono i &quot;fiale strette&quot;. Se si usa fiale con diametro maggiore, si ha la necessità di aumentare il numero delle femmine / maschi da collocare nel flacone in modo che le femmine sarebbero pronti per essere analizzati in 4 - 5 giorni. Mentre il protocollo qui descritto specificamente concentra sull&#39;esame femmine preferenze tra saccarosio e substrati strisciamento, può essere adattato per studiare delle femmine preferenza in altre condizioni. Ad esempio, queste camere possono essere usati per assapreferenza y moscerini nella scelta tra agarosio più duro e morbido (es., 1% vs. 1,5% agarosio), così come substrati che differiscono in altri segnali chemosensoriali (ad es., 3% di acido acetico). Se munito di coperchio che contiene LED per illuminare uno o entrambi i substrati, la camera può anche fornire una piattaforma efficace per condurre studi optogenetic (Figura supplementare 4). Infine, vale la pena notare che, anche se questa configurazione consente un throughput più elevato per analizzare le preferenze deposizione delle uova di Drosophila, ha alcune importanti limitazioni. Innanzitutto, la dimensione fissa delle camere limita la flessibilità del dosaggio comportamentale. Ad esempio, si dovranno fare nuove camere al fine di testare come gli altri parametri (ad es., La distanza tra i supporti) possono influenzare le preferenze di deposizione delle uova di mosche. Inoltre, questo sistema non cattura l&#39;ambiente complesso Drosophila incontro in natura. E &#39;molto raro che vola dovrà decidere tra un puro substrato di saccarosio rispetto a un substrato di pianura puro per la deposizione delle uova in natura. Così, mentre il nostro test è efficace nello studio le basi neurali di un semplice compito decisione, si ha la necessità di essere a conoscenza che le specifiche &quot;decisioni&quot; femmine fanno nelle nostre camere potrebbe non essere etologicamente rilevante.

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors1-3.
Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare and rank their options before "committing" to depositing an egg onto a given option4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.
In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Sucrose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water bath&#160;</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">15-462-6Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LifeCam Cinema webcam</td>
			<td style="width:104px;">Microsoft</td>
			<td style="width:119px;">H5D-00013</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:269px;">Red LEDs</td>
			<td style="width:104px;">Cree</td>
			<td style="width:119px;">C503B-RAN-CA0B0AA1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Egg-laying chambers</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Camera holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LED holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Fly vials (narrow)</td>
			<td style="width:104px;">Genesee</td>
			<td style="width:119px;">32-116BC</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput assay to examine egg-laying preferences of individual drosophila melanogaster

Recentemente, la preferenza deposizione delle uova di Drosophila è emersa come un modello geneticamente trattabili per studiare le basi neurali di semplici processi decisionali. Quando si seleziona siti per depositare le loro uova, mosche femmine sono in grado di rango l&#39;attrattiva relativa delle loro opzioni e scegliere il &quot;maggiore tra due beni.&quot; Tuttavia, la maggior parte dei test di preferenza deposizione delle uova non sono pratici se si vuole prendere un approccio sistematico screening genetico per la ricerca di base del circuito alla base di questo semplice processo decisionale, in quanto sono basati sulla popolazione e laborioso da configurare. Per aumentare il throughput di studiare delle preferenze ovaiole di singole donne, abbiamo sviluppato le camere personalizzate che ciascuno può allo stesso tempo saggio preferenze deposizione delle uova fino a trenta individuale vola così come un protocollo che garantisce ogni femmina ha un alto tasso di deposizione delle uova (in modo che la loro preferenza è facilmente distinguibile e più convincente). Il nostro approccio è semplice da eseguiree produce risultati molto consistenti. Inoltre, queste camere possono essere equipaggiate con diversi accessori per consentire la registrazione video gli animali deposizione delle uova e per fornire luce per gli studi optogenetics. Questo articolo fornisce i progetti per la realizzazione di queste camere e la procedura per preparare le mosche per essere analizzati in queste camere.

Le camere di deposizione delle uova sono composti da diversi pezzi: un pezzo di substrato (in basso), un pezzo di divisione (al centro), un pezzo di carico (in alto), e 2 porte scorrevoli (Figura 1A - D). Questi pezzi sono utilizzati per indipendentemente mosche di impostazione e substrati prima degli esperimenti di deposizione delle uova. La figura 1F mostra come flaconcini devono guardare quando le mosche di sesso femminile sono pronte per la deposizione delle uova. Quando mosche sono date una scelta tra un substrato piano e un substrato contenente saccarosio, femmine robustamente preferito il substrato normale per la deposizione delle uova come mostrato in Figura 2. <img alt="Figura 1" src="/files/ftp_upload/53716/53716fig1.jpg" /> Figura 1. Chambers deposizione delle uova e la deposizione delle uova protocollo. (A) completamente assemblati uovo camera -laying. (B) pezzi smontati. a: il pezzo di carico (in alto), b: lapezzo divisore (al centro), c: il substrato (in basso) pezzo. Disegni tecnici di questi pezzi sono mostrati nella Figura complementare 1 -. 3 (C) Il pezzo caricamento della camera. un &#39;: porte, un &quot;scorrevoli:.. rotaie I fogli di plastica vengono inseriti nel pezzo di carico per servire da pavimento per mantenere le mosche caricati in posizione Tipicamente messo nastri di colore sul bordo dei fogli di plastica (freccia rossa). (D) il substrato (in basso) e il divisore (al centro) pezzi della camera. agarosio viene depositato in singole mangiatoie per servire come substrati di deposizione delle uova (frecce). Quadrilatero delinea l&#39;arena di deposizione delle uova per una mosca. (e ) Giorno 0 di femmine / maschi in una fiala yeasted raccolti (F) 4 ° giorno -.. 5 delle femmine / maschi in una fiala yeasted raccolti notare che larve e adulti femmine hanno mangiato la maggior parte del lievito e la superficie del cibo è diventato occupato da larve. Il cibo superficie bagnata brulicante di larve impedisce femmine di deporre più uova nel flacone. (G) Schema che descrive il protocollo per la creazione di esperimenti comportamentali deposizione delle uova. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig1large.jpg" target="_blank">(Si prega di cliccare qui per vedere una versione più grande di questa figura)</a> <img alt="figura 2" src="/files/ftp_upload/53716/53716fig2.jpg" /> Figura 2. Risultati per mosche scegliere tra saccarosio e substrati Plain rappresentante la deposizione delle uova. (A) Vista laterale dei risultati ovaiole di tipo selvatico vola quando sono stati dati una scelta tra saccarosio (150 mm) e substrati di pianura. Scatola bianca delinea una scena di deposizione delle uova per una mosca. (B) Vista superiore dei risultati di deposizione delle uova. Scatola bianca delinea una scena di deposizione delle uova per una mosca. (C) Indice Preferenza (<html> PI) di tipo selvatico vola quando viene chiesto di scegliere tra un substrato di saccarosio contenente e un substrato pianura. PI per ogni femmina è calcolato come segue: (numero di uova su substrato di saccarosio - il numero di uova su substrati di pianura) / numero totale di uova. Bar errore indica SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig1large.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">(Clicca qui per vedere una versione più grande di questa figura)</a> <img alt="Supplementare Figura 1" src="/files/ftp_upload/53716/53716supfig1.jpg" /> Supplementare Figura S1 ingegneria disegni per il divisore e il pezzo substrato della Camera.. (A - A &quot;) Viste diverse del pezzo substrato della Camera. (B - B &#39;) Diversi punti di vista del pezzo divisorio centro della camera.d / 53716 / Supplemental1.jpg &quot;style =&quot; font-size: 14px; line-height: 28px; &quot;target =&quot; _ blank &quot;&gt; (Si prega di cliccare qui per scaricare questo file) <img alt="Supplemental Figura 2" src="/files/ftp_upload/53716/53716supfig2.jpg" /> Supplementare Figura S2 Disegni tecnici per il carico pezzo della Camera.. (A - A &#39;) diverse vedute dello strato superiore del pezzo di carico della camera. . (B - B &#39;) Diversi punti di vista lo strato inferiore del pezzo di carico della camera <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental2.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">(Clicca qui per scaricare questo file)</a> <img alt="Supplemental Figura 3" src="/files/ftp_upload/53716/53716supfig3.jpg" /> Supplementare Figura S3. <strong&gt; Ingegneria Disegno per il portello scorrevole ed il Rail della Camera. (A) La guida fissa sul pezzo superiore della camera. (B) La porta scorrevole per la camera. ( <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental3.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">Clicca qui per scaricare questo file)</a> <img alt="Supplemental Figura 4" src="/files/ftp_upload/53716/53716supfig4.jpg" /> Supplementare Figura S4. Accessori e personalizzazione delle sezioni di deposizione delle uova. (A) camere di deposizione delle uova dotati di telecamere per registrare video mosche durante gli esperimenti di deposizione delle uova 13. Ctrax 14 con estensione 12 viene utilizzato per tracciare le mosche e personalizzato codice MATLAB è usato per tracciare le traiettorie. (B) il programma di installazione per illuminare substrati di deposizione delle uova con ligh rosso<html> t. I LED (1) sono collegati ad un driver LED (2). intensità LED è controllato dal microcontrollore (3). Per ulteriori informazioni sulla fotocamera, sistema di erogazione di luce e monitoraggio degli animali in condizioni di luce anomale, vedere Zhu et al. e Stern et al. (C) I LED rossi (luce spenta) allegati alla parte superiore delle camere di deposizione delle uova. (D) I LED rossi (acceso) attaccati alla parte superiore delle camere di deposizione delle uova. L&#39;intensità del LED di LED rossi è in genere impostato per essere intorno a 7-10 W / mm quando CsChrimson 15 viene utilizzato per attivare i neuroni. (E) deposizione delle uova camere dotate di telecamere e LED per le mosche registrare video durante esperimenti con la luce rossa (luce spenta). (F) camere di deposizione delle uova dotati di telecamere e LED per le mosche registrare video durante esperimenti con la luce rossa (luce accesa). (t: 28px; &quot;target =&quot; _ blank &quot;&gt; Clicca qui per scaricare questo file)

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput test per esaminare Preferenze deposizione delle uova dei singoli<em&gt; Drosophila melanogaster</em

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

high-throughput-assay-to-examine-egg-laying-preferences-individual

Nanyang Technological University

Research

JoVE Journal

Neuroscience

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

7.9K Views.  Duke University. This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Video: High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ricerca

Neuroscienze

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection associato e test Elettrofisiologia allo schermo per la bioattività dei composti con il Drosophila melanogaster Sistema Fiber Giant

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Metodi per test Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Stimolazione Optogenetic del comportamento in Fuga<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Assay Semplice per la quantificazione della Social Prevenzione in<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

Condizioni che influiscono spazio sociale in<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

Ad alta risoluzione quantificazione del comportamento Odor-guidata<em&gt; Drosophila melanogaster</em&gt; Utilizzo della<em&gt; Flywalk</em&gt; Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Un test comportamentale per Mechanosensation di cloni in marcm-based<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Il capillare alimentatore Assay Misure assunzione di cibo in<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analizzando Synaptic Modulazione di<em&gt; Drosophila melanogaster</em&gt; Fotorecettori dopo l&#39;esposizione alla luce prolungata

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...