Questo lavoro è stato finanziato da una borsa di studio della Stanford Dean (SEJdV), il National Institutes of Pioneer Award Direttore Salute (TRC DP0035350), un premio McKnight Foundation studioso (TRC) e R01 EY022638 (TRC).

1. Genera mosche channelrhodopsin <ol><li> Croce UAS-CHR2 mosche con il driver Gal4 di vostra scelta, usiamo G105-Gal4, che si esprime in foma-1 neuroni nel lobo ottico. </li><li> Per eliminare la possibilità di una risposta alla stimolazione visiva luce blu, entrambe le linee mosca sono in aw sfondo + norpA. </li><li> Risultato finale: w + norpA; G105-Gal4/UAS-ChR2 + </li><li> Dopo adulto vola Eclose, messo femmine selezionati alimenti freschi, integrata con 10 pM tutto-trans-retiniche (un co-fattore necessario per CHR2) e al riparo dalla luce, per 3 giorni prima di eseguire il test del comportamento. </li></ol> 2. Prova del 10 pM All-trans-retinale cibo avanzato <ol><li> Sciogliere 100 mg di tutto-trans-retinale nel 17,6 ml di etanolo al 95%, che il 20 mM retina. Tenere tutto-trans-retinale al riparo dalla luce in ogni momento. </li><li> Melt standard alimentari fly farina di mais in forno a microonde, e lasciare raffreddare fino a caldo al tatto. </li><li> Mescolare 50 ml di 20 mMtutto-trans-retinale in flaconcini da 10 ml di cibo mosca. </li><li> Lasciare raffreddare e conservare fiale al riparo dalla luce. </li></ol> 3. Attrezzatura <ol><li> Puntali: Standard 1.000 puntali ul vengono tagliati vicino alla punta, la creazione di un diametro dei pori di ~ 2,25 millimetri. </li><li> Piattaforma (vedi Figura 1). <ol><li> Una base Delrin, 17 cm x 25 cm, è stato costruito con fori filettati ad ogni angolo per adattarsi ¼ &quot;NPT raccordi del tubo del liquido di raffreddamento. </li><li> Un supporto verticale, realizzato in Delrin, è attaccata al centro della base. Le dimensioni di ingombro sono 25 mm x 40 mm x 65 mm (larghezza x profondità x altezza). Una scanalatura 10 mm di larghezza corre la lunghezza del supporto, con una vite sul fondo. Una piattaforma è fissato alla parte superiore del supporto, 25 mm x 40 mm x 10 mm, con un foro di diametro 3,5 mm allineata con la scanalatura nel supporto. </li></ol></li><li> Array di LED (si veda la Figura 1). <ol><li> Quattro braccia di tubo di raffreddamento, ~ 18 cm di lunghezza, vengono fissate sulla bas piattaformae utilizzando il connettore del tubo del liquido di raffreddamento. Tubo del liquido di raffreddamento viene utilizzato solo come supporto strutturale e non viene utilizzato per il raffreddamento. </li><li> Scanalature distanziate correttamente vengono tagliati nel pezzo di refrigerante finale getti di ciascun braccio di apporre un dissipatore di calore al fine di ogni braccio. </li><li> A Rebel LED blu Tri-Stars è montato ad ogni dissipatore di calore utilizzando pre-taglio del nastro adesivo termico. Un Carclo 18 ° Tri-obiettivo viene apposto su ogni Tri-Star. </li><li> LED Tri-Stars sono collegati ai driver DC BuckPuck e un alimentatore come specificato. Abbiamo organizzato il nostro set-up con l&#39;alimentazione BuckPuck due Tri-Stars in serie. </li><li> Illuminazione di tutti e quattro LED Tri-Stars a 700 mA ha prodotto un irraggiamento di 713 W / m 2 sulla nostra piattaforma. </li></ol></li><li> Camera: La camera è montata su un cavalletto piccola e focalizzata sulla parte superiore della piattaforma. </li></ol> 4. Saggio comportamentale <ol><li> Brevemente anestetizzare mosche su ghiaccio. </li><li> Mettere mosche singoli puntali, con nastro per chiudereentrambe le estremità della punta. </li><li> Dopo che le mosche si sono svegliati e stanno esplorando attivamente la punta della pipetta, rimuovere il nastro e mettere una pipetta nella scanalatura nel supporto verticale. La vite viene utilizzato per fissare la punta della pipetta in posizione e chiudere il fondo della punta. </li><li> Come la mosca esplora la punta della pipetta (in genere 30 - 60 sec), avviare la registrazione della videocamera poco prima al volo emerge dalla punta sulla piattaforma. </li><li> Dopo il cavalcavia è emerso sulla piattaforma, attendere 1-2 seco, e accendere il LED blu. Utilizzare un timer per misurare manualmente il tempo fino al volo avvia volo. </li></ol>

<ol>
	<li>Venken, K., Simpson, J., Bellen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulations+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulations of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Gordon, M., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+in+a+Drosophila+taste+circuit.">Motor control in a Drosophila taste circuit.</a> Neuron. 61, 373-384 (2009).</li><li>Suh, G. S. B., 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=Light+activation+of+an+innate+olfactory+avoidance+response+in+Drosophila.">Light activation of an innate olfactory avoidance response in Drosophila.</a> Current Biology. 17, 905-908 (2007).</li><li>Zhang, W., Ge, W., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+for+light+control+of+Drosophila+behaviors+through+Channelrhodopsin+2-mediated+photoactivation+of+targeted+neurons.">A toolbox for light control of Drosophila behaviors through Channelrhodopsin 2-mediated photoactivation of targeted neurons.</a> European Journal of Neuroscience. 26, 2405-2416 (2007).</li><li>Nagel, 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Science. 100, 13940-13945 (2003).</li><li>de Vries, S., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loom-sensitive+neurons+link+computation+to+action+in+the+Drosophila+visual+system.">Loom-sensitive neurons link computation to action in the Drosophila visual system.</a> Current Biology. 22, 353-362 (2012).</li><li>Card, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+behaviors+in+insects.">Escape behaviors in insects.</a> Current Opinion in Neurobiology. 22, 1-7 (2012).</li><li>Card, G., 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=Visually+mediated+motor+planning+in+the+escape+response+of+Drosophila.">Visually mediated motor planning in the escape response of Drosophila.</a> Current Biology. 18, 1300-1307 (2008).</li><li>Fotowat, H., Fayyazuddin, A., Bellen, H. J., Gabbiani, F. <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+neuronal+pathway+for+visually+guided+escape+in+Drosophila+melanogaster.">A novel neuronal pathway for visually guided escape in Drosophila melanogaster.</a> Journal of Neurophysiology. 102, 875-885 (2009).</li><li>Card, G., 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=Performance+trade-offs+in+the+flight+initiation+of+Drosophila+melanogaster.">Performance trade-offs in the flight initiation of Drosophila melanogaster.</a> The Journal of Experimental Biology. 211, 341-353 (2008).</li><li>Hammond, S., O'Shea, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+flight+initiation+in+the+fly.">Escape flight initiation in the fly.</a> Journal of Comparative Phsyiology A. 193, 471-476 (2007).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> PNAS. 58, 1112-1119 (1967).</li><li>Bloomquist, B., 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=Isolation+of+a+putative+phospholipase+C+gene+of+Drosophila,+norpA,+and+its+role+in+phototransduction.">Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.</a> Cell. 54, 723-733 (1988).</li><li>Gohl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+in+vivo+system+for+directed+dissection+of+gene+expression+patterns.">A versatile in vivo system for directed dissection of gene expression patterns.</a> Nature Methods. 8, 231-237 (2011).</li><li>Zhang, F., 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=Red-shifted+optogenetic+excitation:+a+tool+for+fast+neural+control+derived+from+Volvox+cateri.">Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox cateri.</a> Nature Neuroscience. 11, 631-633 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

<html> Abbiamo dimostrato la stimolazione optogenetic di comportamenti di fuga da bagno liberamente a piedi mosche in brillante luce blu. Questo approccio può essere facilmente adattato ad esaminare altri comportamenti nelle mosche liberamente a piedi, e può essere scalata a grandi piattaforme semplicemente le piastrelle array di LED che abbiamo usato su un&#39;area più grande. Utilizzando la telecamera a basso costo descriviamo, o altri sistemi di telecamere disponibili, l&#39;utente può personalizzare il frame rate...

Un arsenale crescente di strumenti geneticamente codificati sono stati sviluppati per manipolare attività neurale in cellule specifiche in Drosophila melanogaster 1. Questi strumenti consentono l&#39;attivazione non invasiva o silenziamento di neuroni specifici nel intatta di un animale e liberi di muoversi. Tra questi, Channelrhodopsin2 (CHR2), un fotoattivabile canale cationico, offre vantaggi, poiché può essere controllata temporalmente e rapidamente indotta. Quando i neuroni che esprimono CHR2 esposti a brillante blu (470 nm), luce che rapidamente depolarizzare e presentano elevati tassi di cottura 3-5. Tale attivazione mirata dei neuroni specifici negli animali liberi di muoversi ha rivelato la sufficienza dei neuroni particolari per comportamenti come la CO 2 evitamento 3, estensione 2,4 proboscide, e gigante in fibra di startle risposte mediate 4. Tuttavia, poiché le sorgenti luminose intense necessarie per stimolare CHR2 anche stimolare fotorecettori, applicando optecniche togenetic al sistema visivo è stato limitato. Combinando un approccio optogenetic con una mutazione che altera fototrasduzione, abbiamo dimostrato che l&#39;attivazione di uno specifico cluster di neuroni nel lobo ottica della mosca può guidare il comportamento di fuga utilizzata per evitare la collisione 6. La maggior parte, se non tutti, gli animali visivi presentano un comportamento di fuga per evitare collisioni con oggetti che sopraggiungono. Camminare o stazionario mosche, quando sono presentati con una collisione incombente, decollo in volo, lontano dalla collisione imminente 7-9. I decolli sono caratterizzati da ali alzate prima del decollo e una traiettoria di volo instabile 10,11. Questa risposta è distinto da gigante in fibra di risposta mediata trasalimento, salti che non sono preceduti da ali alzate, e di solito portare a una caduta in caduta libera 4,9. Dopo aver individuato un gruppo specifico di neuroni sensibili telaio nel lobo ottico, Foma-1 neuroni, che sono uniquely sintonizzati per codificare gli oggetti che si avvicinano, abbiamo cercato di sondare il loro coinvolgimento nel comportamento telaio della mosca fuga. Qui mostriamo l&#39;uso di optogenetics per attivare selettivamente questi neuroni, da attuarsi con il comportamento di fuga della mosca. Usiamo il Gal4-UAS sistema attivatore trascrizionale per guidare l&#39;espressione di CHR2-1 nei neuroni FOMA. CHR2 richiede il cofattore tutto-trans-retinica e come questo si trova in bassi livelli del sistema nervoso centrale Drosophila deve essere integrato nella dieta delle mosche. 3,4 Come luce è utilizzato per attivare CHR2 e vola mostrare comportamenti phototactic forti 12, si è cercato di eliminare la possibilità di una risposta allo stimolo visivo. Per fare questo, abbiamo utilizzato gli animali che erano mutante omozigote per un allele nullo del gene norpA, che codifica per un componente critico della fototrasduzione a cascata, fosfolipasi C-β. Fotorecettori in tali mosche mutanti sono in grado di responsabilitàd alla luce 13. Per testare la stimolazione optogenetic della risposta di fuga, abbiamo bisogno di isolare una sola mosca e fare il bagno in brillante luce blu. Per fare questo, abbiamo posto le mosche singole puntali. Una punta della pipetta è posto in un supporto personalizzato, in modo tale che la mosca si geotactically a piedi fino alla punta e fuori su una piattaforma rettangolare. La mosca è in grado di camminare liberamente sulla parte superiore di questa piattaforma. La piattaforma è circondata da quattro matrici LED blu, contenenti ciascuno 3 LED, focalizzati sulla parte superiore della piattaforma. Dopo la mosca è sulla piattaforma, i LED sono illuminati, e la risposta della mosca è registrati con un telecamera ad alta velocità 6.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> All-trans retinico </td><td> Advance Scientific &amp; Chemical Inc </td><td> R3041 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Dissipatore di calore 9,2 C / W </td><td> Luxeonstar </td><td> LPD30-30B </td><td> 30 mm quadrati X 30 mm </td></tr><tr><td> Carclo 18 ° Tri-Lens </td><td> Luxeonstar </td><td> 10507 </td><td></td></tr><tr><td> Rebel LED blu su Tri-Star Base </td><td> Luxeonstar </td><td> MR-B0030-20T </td><td> 470 nm, 174 lm @ 700 mA. </td></tr><tr><td> 700 mA DC BuckPuck driver </td><td> Luxeonstar </td><td> 3021-DE-700 </td><td></td></tr><tr><td> Cablaggio per il driver BuckPuck </td><td> Luxeonstar </td><td> 3021-HE </td><td></td></tr><tr><td> Pre-taglio del nastro adesivo termico </td><td> Luxeonstar </td><td> LXT-S-12 </td><td> 20 Base mm Hex </td></tr><tr><td> Snap-Loc tubo del liquido di raffreddamento, ¼ &quot;ID </td><td> McMaster-Carr </td><td> 5307K49 </td><td></td></tr><tr><td> Snap-Loc Connettore tubo del liquido di raffreddamento </td><td> McMaster-Carr </td><td> 5307K39 </td><td> ¼ &quot;NPT maschio </td></tr><tr><td> Laboratorio di grado Switching programmabile Alimentazione DC </td><td> BK Precision </td><td> 1698 </td><td></td></tr><tr><td> Exilim fotocamera </td><td> Casio </td><td> EX-FH20 </td><td></td></tr></tbody></table>

optogenetic stimulation of escape behavior in drosophila melanogaster

Un numero crescente di strumenti geneticamente codificati sono sempre a disposizione che permettono la manipolazione non invasiva dell&#39;attività neurale dei neuroni specifici in Drosophila melanogaster 1. Primo fra questi sono strumenti optogenetic, che consentono l&#39;attivazione o silenziamento dei neuroni specifici nel intatta di un animale e muoversi liberamente con la luce brillante. Channelrhodopsin (CHR2) è un fotoattivabile canale cationico che, attivato dalla luce blu, provoca depolarizzazione dei neuroni che lo esprimono. CHR2 è stato efficace per identificare i neuroni critiche per comportamenti specifici, come ad esempio evitare di CO 2, l&#39;estensione proboscide e gigante in fibra di risposte di allarme mediata 2-4. Tuttavia, poiché le sorgenti luminose utilizzate per stimolare CHR2 anche stimolare fotorecettori, queste tecniche optogenetic non sono stati precedentemente utilizzati nel sistema visivo. Qui, uniamo un approccio optogenetic con una mutazione che altera fototrasduzione a demonstrate che l&#39;attivazione di un gruppo di telaio sensibili neuroni nel lobo ottico della mosca, FOMA-1 neuroni, può guidare un comportamento di fuga utilizzato per evitare la collisione. Abbiamo utilizzato un allele nullo di un componente critico della cascata phototransduction, fosfolipasi C-β, codificata dal gene norpA, per rendere l&#39;aria cieco e anche usare la Gal4-UAS sistema attivatore trascrizionale per guidare l&#39;espressione del CHR2 in Foma-1 neuroni. Mosche individuali sono posto su una piccola piattaforma circondata da LED blu. Quando i LED sono illuminati, le mosche rapidamente decollo in volo, in un modo simile a quello visivo guidato telaio-escape comportamento. Riteniamo che questa tecnica può essere facilmente adattato per esaminare altri comportamenti liberi di muoversi in mosche.

Cieco vola che ha espresso CHR2 o il G105 solo conducente mostrano un basso tasso di decollo in seguito alla loro illuminazione con la luce blu. Cieco vola esposto lo stesso tasso di decollo senza illuminazione (Figura 2), suggerendo che i decolli erano spontanei, piuttosto che a causa della illuminazione con luce blu. Quando il CHR2 è espresso nelle FOMA1 neuroni, tuttavia, illuminazione con luce blu suscita la risposta di fuga. Oltre il 50% delle mosche testati tolse entro 1 secondo di illuminazione,...

Watch this Scientific Journal Video about Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster at JoVE.com

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Strumenti optogenetic geneticamente codificati consentono la manipolazione non invasiva dei neuroni specifici del<em&gt; Drosophila</em&gt; Cervello. Tali strumenti in grado di identificare i neuroni la cui attivazione è sufficiente per scatenare o sopprimere comportamenti particolari. Qui vi presentiamo un metodo per l&#39;attivazione Channelrhodopsin2 che si esprime nei neuroni mirate a mosche liberamente a piedi.

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 ...

optogenetic-stimulation-of-escape-behavior-in-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

17.3K Views.  Stanford University. Strumenti optogenetic geneticamente codificati consentono la manipolazione non invasiva dei neuroni specifici del Drosophila Cervello. Tali strumenti in grado di identificare i neuroni la cui attivazione è sufficiente per scatenare o sopprimere comportamenti particolari. Qui vi presentiamo un metodo per l'attivazione Channelrhodopsin2 che si esprime nei neuroni mirate a mosche liberamente a piedi.

Video: Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Registrazioni Sensillum singolo in Insetti Drosophila melanogaster E Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Ricerca

Neuroscienze

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La registrazione simultanea dei segnali di calcio da parte dei neuroni identificati e comportamento alimentare di Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Determinazione del locomotore attività spontanea in<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Stimolazione optogenetic del nervo uditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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 Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Un modo semplice per misurare alterazioni in ricompensa il comportamento di ricerca Utilizzando<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Quantificazione del comportamento del grooming di Drosophila

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetica trascinamenti di teta Hippocampal oscillazioni nel comportarsi topi

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...