Ringraziamo Sarah Wu e Camille Graham per lo sviluppo di prime fasi del saggio freddo della sonda, il Bloomington Drosophila Stock Center per gli stock Fly, ed i membri del laboratorio Galko per la lettura critica del manoscritto. Questo lavoro è stato sostenuto da NIH NRSA (NIH F31NS083306) per HNT, e dal NIH R01NS069828, R21NS087360 e Università del Texas MD Anderson Clark Fellowship nella ricerca di base a MJG.

1. Preparazione di larve <ol><li> Sollevare azioni o incroci genetici in un incubatore a 25 ° C. <ol><li> Se la coltura di una croce, utilizzare 20-25 femmine vergini e 15-20 maschi per flaconcino contenente regolare dei media farina di mais mosca. Consentire femmine depongono le uova per circa 48 ore prima di trasferirli ad un nuovo flacone di cibo. </li></ol></li><li> 4-5 giorni dopo lay uovo, raccogliere 3 ° larve instar del genotipo desiderato spruzzando delicatamente un flusso d&#39;acqua nel cibo incurvato e larve, e versare il contenuto in una scatola di Petri pulito medie (60 mm x 15 mm). </li><li> Successivamente, ordinare delicatamente larve formato usando pinze o un pennello per precludere maggiore &quot;vagare 3 ° instar&quot; o piccole larve malato di essere testato. Scartare larve contenente eventuali marcatori genetici indesiderate o di bilanciamento. </li><li> Avere un compagno di piatti a base di etichette membri laboratorio o contenitori in cui le larve ordinato verrà posizionato in modo che lo sperimentatore può essere &quot;cieco&quot; per la excondizione Perimental o il genotipo delle larve, se applicabile. Lo sperimentatore può applicare etichette decodificati i dati raccolti dopo l&#39;esperimento utilizzando una chiave generata da un laboratorio-membro. </li><li> Utilizzando uno scoopula, trasferire una monetina piccola quantità di alimenti freschi in (35 mm x 10 mm o 60 mm x 15 mm) etichettata e pulita piastra di Petri riempita a metà strada con acqua a temperatura ambiente. </li><li> Spostare delicatamente le larve ordinato sul cibo (per evitare la fame o disidratazione se il test è previsto per prendere più di 20 minuti) con pinze o pennello. </li></ol> 2. freddo Probe Assay <ol><li> Accendere l&#39;unità sonda di freddo che permette a pochi minuti che si raffreddi alla temperatura desiderata. Se l&#39;impostazione a temperature più basse, la formazione di condensa è probabile che si formano lungo la sonda. </li><li> Eliminare l&#39;umidità in eccesso con un laboratorio wipe prima dell&#39;applicazione per la larva. Quando non è in uso immediato, posto isolante tappo sulla sonda sia isolare e per mantenere la punta pulita e prevenire danni. </li><li> Posizionare una metà 3 ° larva su un pezzo sottile di vinile mobile scuro (tipicamente, utilizzare un piccolo pezzo tagliato da un legante notebook) sotto un microscopio a campo luminoso. Gli aiuti di vinile nero a contrasto e la visualizzazione in movimento la larva senza toccarlo per allinearlo correttamente con la sonda. </li><li> Regolare il microscopio (vedi Tabella Materiali) e parabola (vedi Tabella Materiali) a luminosità media (50-75% luminosità max) per fornire contrasto ed impedire la larva si secchi troppo rapidamente. </li><li> Eliminare qualsiasi larve che non prima esposizione normale locomozione peristaltica come potrebbero confondere i risultati. La larva dovrebbe essere umido dalla piastra di Petri di acqua altrimenti la larva si attacchi al vinile inibizione normale locomozione. L&#39;acqua non deve pozza intorno alla larva. </li><li> Avanzare la sonda manualmente verso la larva ad un angolo di 90 ° rispetto all&#39;asse del corpo anteroposteriore, utilizzando il vinile per spostare la larva in corposizione rect (vedere Figura 1A). </li><li> Orientare la punta della sonda per gettare delicatamente sulla superficie dorsale metà della larva con la sonda ad un angolo di circa 45 ° alla fase microscopio. </li><li> In caso di contatto della sonda, avviare un timer di laboratorio. Applicare abbastanza pressione verso il basso per far rientrare leggermente la superficie della cuticola larvale pur consentendo avanti o arretramento. </li><li> Tenere la sonda in posizione per un massimo di 10 s o fino a quando si osserva una risposta comportamentale evocata freddo - a seconda di quale si verifica prima. Quindi rimuovere la sonda. </li><li> Registrare la risposta comportamentale osservato e la latenza di risposta. Le larve che non risponde entro 10 s sono considerati &quot;non-responder&quot;. La latenza può essere registrato anche per responder e utilizzati per misurare cambiamenti nella risposta robustezza / ampiezza. </li><li> Eliminare larva e preparare il prossimo. </li><li> Ripetere i passaggi 2,3-2,11 fino a raggiungere il numero desiderato di test di larve (tre serie di n = 20-40 larve erano used qui). </li></ol>

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Una domanda di brevetto negli Stati Uniti (CL) è in corso sulla progettazione della sonda fredda. Ulteriori informazioni sui dettagli dei numeri di parte della sonda fredda e la costruzione, nonché la consulenza per la realizzazione di strumento verranno forniti su richiesta.

Il test qui descritto può essere utilizzato per valutare qualitativamente e quantitativamente nocicezione o sensibilizzazione nocicettiva in larve di diversi background genetici, influenze ambientali, e / o le condizioni di danno-indotta. Poiché questo test consente di applicare focale di uno stimolo freddo, con questo strumento si può valutare la funzione di un sottoinsieme di neuroni sensoriali periferici specificamente in risposta alle basse temperature. È interessante notare che questi comportamenti evocati fred...

Drosophila ha dimostrato di essere molto utile per l&#39;identificazione di nuovi geni conservati e circuiti neuronali che sottendono comportamenti complessi. Mosche forniscono un sofisticato toolkit genetico e un sistema nervoso semplificato che permettono precisa manipolazione genetica e neuronale 1, 2, 3, 4 per sezionare le basi cellulari e molecolari della nocicezione 5, 6, 7. Larve sono particolarmente utili per queste analisi, dato che saggi comportamentali per tocco delicato 8, 9, 10, 11 calore nocive, 12, 13 e sensazione meccanica di stimoli nocivi 4, </sup&gt; 11 sono già stati stabiliti, e la cuticola larvale trasparente permette l&#39;imaging dal vivo o fisso dell&#39;epidermide e neuroni sensoriali sottostanti. Recentemente, un saggio per freddo nocivo è stato anche sviluppato 7, che descriveremo in maggior dettaglio qui. Utilizzando una multa sonda fredda, conico-punta, mostriamo che Drosophila larve presentano un insieme di comportamenti reattivi specifici a freddo, distinto da comportamenti osservati durante la locomozione normale, seguito tocco delicato, o dopo duro stimolo meccanico temperatura o alta 7, 8, 11 . I comportamenti specifici a freddo includono un robusto contrazione tutto il corpo (CT), una 45-90º aumento dei segmenti posteriori (PR) e un aumento simultaneo del segmenti anteriore e posteriore in una forma ad U (US). La prevalenza di questi comportamenti aumenta con temperature decrescenti ma ciascuno picchi sleggermente diverse temperature fredde. Lavori recenti suggeriscono che le risposte CT sono mediati da diversi neuroni sensoriali periferici rispetto a quelle che rispondono a calore nocive o dura stimoli meccanici 7. Proprio come nocicettori vertebrati, Drosophila dendritiche multipla (md) neuroni sensoriali periferici hanno strutture dendritiche complesse che arborize oltre l&#39;epidermide 1. neuroni md sono presenti in ogni segmento corporeo larvale, proiettando loro assoni al cordone nervoso ventrale 14. neuroni sensoriali md sono suddivisi in quattro classi differenti (I-IV) basati sulla morfologia dendritica e hanno diverse funzioni sensoriali 4, 9, 10, 15, 16, 17. Mentre i neuroni classe IV sono necessari per larvali risposte rollio lateralea temperature elevate o rigide stimoli meccanici 4, classe III neuroni sono necessari per delicato tocco risposte 9, 10 e non solo sono attivati dal freddo, ma sono necessarie anche per le risposte comportamentali evocati freddo 7. Entrambi i neuroni classe III e IV utilizzano potenziali canali discreti transient receptor (TRP) per facilitare risposte comportamentali ai nociva 7, 11, 18 e stimoli 9, 10, 17, 19 non-nocivo. Inoltre, nocicezione larvale è sensibilizzato infortunio seguito, a livello cellulare 20 e comportamentali 12, 21. Il saggio descritto qui consente la quantification di una normale o potenzialmente alterato risposte comportamentali a basse temperature che variano da nocivi freddo (≤ 10 ° C), freschi innocui (11-17 ° C), a temperatura ambiente (18-22 ° C). Le temperature fredde utilizzati in questo test è in grado di attivare direttamente classe III neuroni sensoriali, suscitando robusti, aumenta calcio riproducibili e risposte comportamentali evocati freddo, che possono essere qualitativamente e quantitativamente analizzati 7. Questo test può essere applicato a larve di qualsiasi genotipo nonché di larve esposto a diverse condizioni ambientali (alimentazione alterato, lesioni, agenti farmacologici) per determinare i fattori sia genetici e ambientali che hanno un impatto nocicezione freddo, la sensibilizzazione nocicettiva e la plasticità nocicettivo. Dato che thermosensation è onnipresente in molte specie, questo test fornisce un valido strumento per lo studio della nocicezione e può scoprire bersagli gene nuovi o interazioni neuronali che migliorerannola nostra comprensione della nocicezione vertebrati. La sonda a freddo su misura (vedi sonda fredda, Table of Materials) utilizza una temperatura controllata ad anello chiuso dispositivo Peltier, che raffredda l&#39;albero alluminio e punta conica per conduzione termica. Un termistore è incorporato all&#39;interno della punta conica alluminio riporta la temperatura in tempo reale sulla centralina. Un dissipatore di calore e la ventola sono fissati al modulo termoelettrico per regolare il carico del effetto Peltier calore (Qc) e quindi l&#39;intervallo di temperatura desiderato (22-0 ° C) può essere realizzato (vedi Unità di controllo termico, Table of Materials). Lo stimolo nocivo freddo della punta della sonda freddo viene applicato a mano alla linea mediana dorsale, a segmento (s) equidistante dalle estremità anteriori e posteriori (circa segmento A4, vedere Figura 1A) della larva. In risposta a stimoli freddi, larve generalmente producono uno dei tre comportamenti evocati freddo a 10 s cut-off: un corpo pienocontrazione (CT), un aumento di 45-90º segmento anteriore e posteriore in una forma ad U (USA), oppure un aumento dei segmenti posteriori (PR) (descritto in Risultati). Nessuno di questi comportamenti vengono eseguite durante la normale locomozione peristaltica o comportamento di foraggiamento. Questi comportamenti sono anche distinti da risposte tocco delicato e la risposta di laminazione avversione ad alte temperature o nocivi stimoli meccanici.

<table><tbody><tr><td>Cold Probe</td><td>Pro-Dev Engineering</td><td>Custom-built on demand</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Thermal Control Unit</td><td>TE Technology</td><td>Custom Built enclosure</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Zeiss Stemi 2000 microscope</td><td>Zeiss</td><td>NT55-605</td><td></td></tr><tr><td>Fiber-Lite MI-150 High Intensity Illuminator</td><td>Dolan-Jenner Industries.</td><td>A20500</td><td></td></tr><tr><td>Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td><td>Schott North America, Inc.</td><td>Schott A08575</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>FS-1670</td><td>Used to sort and handle larvae. Be sure to smooth and blunt forceps tips slightly to lower the risk of accidently puncturing or injuring the larvae</td></tr><tr><td>Paintbrush</td><td>Dick Blick Art Materials</td><td>06762-1002</td><td>Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td></tr><tr><td>35 mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>60mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Piece of black vinyl (at least 2 inches x 2 inches)</td><td></td><td></td><td>Used to provide contrast and orient larvae to the cold probe</td></tr><tr><td>Fisherbrand Scoopula Spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td>Used to move food</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>06-666A</td><td>Used to dry the larvae and cold probe if there is excess moisture</td></tr></tbody></table>

novel assay for cold nociception in drosophila larvae

Come gli organismi di percepire e reagire a temperature nocivi è ancora poco conosciuta. Inoltre, i meccanismi sottostanti sensibilizzazione della macchina sensoriale, come nei pazienti con neuropatia periferica o sensibilizzazione lesioni indotte, non sono ben caratterizzati. Il modello Drosophila geneticamente trattabili è stato utilizzato per studiare le cellule e geni necessari per il rilevamento calore nocive, che ha dato più geni conservati di interesse. Poco si sa tuttavia sulle cellule e recettori importanti per il rilevamento freddo nocivi. Anche se, Drosophila non sopravvive l&#39;esposizione prolungata a temperature fredde (≤10 ° C), ed eviterà fresco, preferendo le temperature più calde in saggi preferenze comportamentali, come essi percepiscono e possibilmente evitare stimoli freddi nocivi è stato recentemente indagato solo. Qui si descrive e caratterizzare i primi freddi nocive (≤10 ° C) del test comportamentaleDrosophila. Con questo strumento e il dosaggio, mostriamo un investigatore come valutare qualitativamente e quantitativamente i comportamenti nocicettivi freddi. Questo può essere fatto in condizioni di coltura normali / sane, o presumibilmente nel contesto della malattia, infortunio o sensibilizzazione. Inoltre, questo test può essere applicato a larve selezionato per genotipi desiderati, che potrebbero avere un impatto thermosensation, dolore, o sensibilizzazione nocicettiva. Dato che il dolore è un processo altamente conservato, da questo test per studiare ulteriormente nocicezione termica sarà probabilmente raccogliere importanti comprensione dei processi di dolore in altre specie, tra cui vertebrati.

Drosophila larve movimento con un movimento peristaltico che comprende pause occasionali, curve testa, e cambiamenti di direzione 22. In risposta alla domanda focale di uno stimolo nocivo freddo tuttavia, larve presentano un insieme di comportamenti unici, a differenza del rullo laterale aversive di calore nocive e stimoli meccanici. Questi comportamenti sono anche diversi dalle risposte al tocco delicato 8, <sup class="...

Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

Novel Assay for Cold Nociception in Drosophila Larvae

Qui mostriamo un nuovo metodo di analisi per studiare nocicezione freddo in Drosophila larve. Questo dosaggio utilizza una sonda custom-built Peltier in grado di applicare una focale stimolo freddo nociva e si traduce in comportamenti specifici a freddo quantificabili. Questa tecnica consentirà un&#39;ulteriore dissezione cellulari e molecolari della nocicezione freddo.

Nuovo metodo di analisi per Cold nocicezione in<em&gt; Drosophila</em&gt; Le larve

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

novel-assay-for-cold-nociception-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

Novel Assay for Cold Nociception in Drosophila Larvae

7.7K Views.  UT MD Anderson Cancer Center.  Qui mostriamo un nuovo metodo di analisi per studiare nocicezione freddo in Drosophila larve. Questo dosaggio utilizza una sonda custom-built Peltier in grado di applicare una focale stimolo freddo nociva e si traduce in comportamenti specifici a freddo quantificabili. Questa tecnica consentirà un'ulteriore dissezione cellulari e molecolari della nocicezione freddo. 

Video: Novel Assay for Cold Nociception in Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Metodi locale e globale valutazione nocicezione termica in<em&gt; Drosophila</em&gt; Le larve

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Ricerca

Neuroscienze

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualizzazione dei Propriorecettori in<em&gt; Drosophila</em&gt; Larve e pupe

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Immunologia e infezioni

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Appetitivo apprendimento associativo olfattiva in<em&gt; Drosophila</em&gt; Le larve

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato<em&gt; Drosophila</em&gt; Larve

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

A Single-fly saggio per comportamento alimentare in<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Un saggio e strumenti migliorati per misurare la nocicezione meccanica nelle larve di Drosophila

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Comportamento

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Un'analisi di gradiente di temperatura per determinare le preferenze termiche delle larve di Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Saggi comportamentali per la manipolazione optogenetica dei circuiti neurali in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Source: Chattopadhyay, A., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

Saggio con sonda termica locale: un metodo per valutare la nocicezione delle alte temperature nelle larve di Drosophila

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

Nuovo metodo di analisi per Cold nocicezione in

Riepilogo

Esplora Altri Video

Metodi locale e globale valutazione nocicezione termica in

Visualizzazione dei Propriorecettori in

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Appetitivo apprendimento associativo olfattiva in

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato

A Single-fly saggio per comportamento alimentare in

Un saggio e strumenti migliorati per misurare la nocicezione meccanica nelle larve di Drosophila

Un'analisi di gradiente di temperatura per determinare le preferenze termiche delle larve di Drosophila

Saggi comportamentali per la manipolazione optogenetica dei circuiti neurali in Drosophila melanogaster

Saggio con sonda termica locale: un metodo per valutare la nocicezione delle alte temperature nelle larve di Drosophila