Vorremmo ringraziare Astha Maltare per generare i dati crawling larvali. Vorremmo ringraziare il Dott. Nicholas Lanson Jr. per dare i suoi commenti sul manoscritto. Questo lavoro è stato sostenuto dal Centro Robert Packard per la SLA presso la Johns Hopkins (a UBP) e la sclerosi laterale amiotrofica Association (UBP) e R01MH083689 dal National Institutes of Mental Health (CDN).

A. larvale Crawling Assay 1. Le larve Collection <ol><li> Impostare un 8 once bottiglia di mosche (10-15 maschi + femmine 10-15). </li><li> Lasciate che le mosche depongono le uova per 24 ore, poi bottiglia trasparente di mosche. (Trasferire gli adulti in una nuova bottiglia e ripetere se necessario). </li><li> Incubare bottiglia per 3-4 giorni, o fino terzo stadio larve sono visibili. </li><li> Aggiungere 50 - 100 ml di 20% saccarosio alla bottiglia con larve e lasciate riposare per 20 minuti. Le larve saranno a galla. </li><li> Raccogliere le larve con una pipetta 25 ml sierologico con la punta tagliata, e posto in un cesto in rete. </li><li> Lavare larve nel cesto maglia due volte con acqua deionizzata H 2 O. Le larve sono ora pronti per gli esperimenti. </li></ol> 2. Per il trattamento di larve con la droga <ol><li> Utilizzare un pennello per trasportare il numero desiderato di larve in un becher 5 ml contenente una soluzione al 5% di saccarosio + farmaco. </li><li> Lasciate che le larve si nutrono per almeno 15 minuti.</li><li> Versare droga trattata con larve in un cesto in rete e risciacquare. Ora sono pronto per l&#39;uso. </li></ol> 3. Assay Locomotore (misurando la distanza totale percorsa o contrazioni parete del corpo) <ol><li> Utilizzare un pennello per il trasporto individuale ad una larva: <ol><li> 15 centimetri capsula Petri contenente il 2% agarosio (precedentemente versato e lasciato indurire) su carta millimetrata di cm 0,2 2 griglia. <ol><li> Contare il numero di linee della griglia incrociate in 1 minuto. </li></ol></li><li> Beh dissezione di un piatto di vetro contenente una soluzione diluita di pasta di lievito. <ol><li> Contare le contrazioni peristalsi (anteriore posteriore completo di movimento = 1 contrazione) in un minuto, osservando al microscopio di dissezione. </li></ol></li></ol></li><li> Ripetere fino a quando i numeri desiderati delle larve sono stati contati. </li></ol> B. Rapid iterativo geotassi negativi (RING) Protocol Questo saggio è stato originariamente disinstallatidescritto dal Gargano et al 5. <ol><li> Raccogliere appena emerse le mosche maschio adulto sotto la luce CO 2 anestesia e il luogo di un alimento standard contenente flacone (o cibo + farmaci). </li><li> Mantenere le mosche a temperatura ambiente (in alto panchina. ~ 22 ° C) per 2-3 giorni per consentire il recupero di CO 2 (e l&#39;accumulo di steady-state i livelli di farmaco se del caso). </li><li> Trasferire circa 25 mosche, senza anestesia in fiale polistirolo preparati. </li><li> Assemblare flaconcini con mosche nell&#39;apparecchiatura ANELLO (Figura 1). </li><li> Lasciare mosche di acclimatarsi all&#39;ambiente, indisturbato, per 15-20 minuti. </li><li> Durante questa fotocamera digitale in tempo luogo ~ 1 m di fronte dell&#39;apparecchio (su una piattaforma, se necessario, per allineare il centro della lente a metà altezza del fiale), mettere a fuoco e zoom la fotocamera sulla apparato, e impostare un timer per 3.0 secondi. </li><li> Cura prendere in mano l&#39;apparato ANELLO con la mano sinistra in modo da non disturb le mosche, e tengono il timer con la mano destra. </li><li> Una drastica toccare l&#39;apparecchio verso il basso sulla superficie del banco di tre volte, assicurando che il rubinetto è difficile da abbattere tutte le mosche al fondo dei flaconi. </li><li> Contemporaneamente con il completamento del terzo rubinetto, inizia il conto alla rovescia 3 secondi. </li><li> A tre secondi scatta una foto. </li><li> Ripristinare il timer per 1 minuto e iniziare. Durante questo tempo ripristinare la fotocamera e mettere a fuoco l&#39;apparato, e impostare un altro canale del timer per tre secondi. </li><li> Dopo 1 minuto, ripetere i passaggi da 1,7-1,10 </li><li> Dopo un totale di 5-6 prove, caricare le immagini su un computer e utilizzare il vostro preferito visualizzatore di immagini per aprire, e segnare l&#39;altezza media si arrampicò per ogni flacone. </li><li> Effettuare analisi statistiche sui vostri gruppi diversi a confronto l&#39;altezza media salito. </li></ol> C. corteggiamento e di accoppiamento test <ol><li> La prima cosa al mattino, ben chiari che producono bottiglie di FLIES da utilizzare. </li><li> Nel corso della giornata (ogni 3-4 ore), la raccolta di recente è emerso maschi sessualmente ingenui e femmine: <ol><li> Mettere i maschi singolarmente in fiale o provette con terreno. </li><li> Mettere 5-6 femmine insieme per flacone / provetta. </li><li> Isolare mosche raccolti a 25 ° C sotto i 12 hr luce / buio per 5 giorni. </li><li> Trasferire uno femmina nella camera di una ruota di accoppiamento. </li><li> Trasferire un maschio nella camera di una ruota di accoppiamento. </li><li> Osservare coppia sotto un microscopio per dissezione i seguenti comportamenti: <ol><li> Orientamento (orienta maschio verso la femmina) </li><li> Tapping (il maschio batte la femmina) </li><li> Canzone Wing (il maschio si estende e vibra un&#39;ala) </li><li> Licking (i maschi lecca i genitali femminili) </li><li> Curling (il maschio si arriccia suo addome sotto se stesso) </li><li> L&#39;accoppiamento tentativo (Leggendo l&#39;attività durante il tentativo di montare la femmina) </li></ol></li></ol></li><li> Osservare per 10 minuti o fino copulazione successo, rilevando il tempo in cui ogni comportamento si verifica (latenza), il tempo totale impegnata in corteggiamento fino accoppiamento (per calcolare l&#39;indice corteggiamento), nonché il numero di coppie di eseguire correttamente un dato comportamento ( frequenza). 100% delle coppie di tipo selvatico generalmente accoppiano entro 5 minuti. </li><li> Calcolare un indice corteggiamento (CI) dividendo il tempo trascorso in corteggiamento diviso per il tempo totale fino copulazione. Per le coppie di questo tipo selvatico varia di solito tra i 0,6-0,8. </li></ol> D. Risultati rappresentativi Crawling test Normale larva di tipo selvatico vagheranno ~ 3 cm / minuto, e presentano ~~~HEAD=NNS 40-50 contrazioni del corpo a parete in un minuto. Abbiamo recentemente sviluppato un modello Drosophila di FUS / TLS-correlato sclerosi laterale amiotrofica che mostra larvale difetto di scansione, durata di vita ridotta e adulti salendo impairment 6. Avevamo l&#39;obiettivo di espressione of wild type e forme mutanti di FUS / TLS per i motoneuroni (OK-371-GAL4 driver) ed eseguito un test di scansione larvale. Come mostrato di seguito, le larve di tipo selvaggio strisciare fino a 12 cm, mentre l&#39;espressione del FUS di tipo selvatico diminuita la capacità delle larve strisciando a circa 6 cm. Gli animali che esprimono il ALS-R521C causando mutazione FUS / TLS dimostrare un danno molto grave nel loro movimento crawling (Figura 1), la scansione solo circa 1 cm al minuto. Negative geotassi test ANELLO Giovani wild-type mosche adulte devono avere un&#39;altezza media salendo di ~ 4-5 cm in hotel 3 secondo periodo di tempo (il tempo può essere regolato da 3 secondi per ospitare diversi ceppi o livelli di attività per definire una determinata altezza media per un dato ceppo / trattamento). Mosche che rimangono sul fondo viene assegnato un valore di 0. Non si consiglia di utilizzare più di 25 mosche per flaconcino, perché allora diventa difficile determinare la posizione di ogni singolo volo per misurare l&#39;altezza. Ndesensibilizzazione o è stata osservata fino alle 6 prove consecutive distanziate 1 minuto a parte che abbiamo impiegato. È critico non riutilizzare i flaconi prova polistirene in questo saggio, dopo gli insiemi iniziali di dati sono raccolti perché le mosche nuovi collocati in fiale utilizzati non salirà nella stessa misura in fiale freschi. <img alt="Figura 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> Figura 1. Di installazione per il saggio RING. La fotocamera digitale è posto ~ 1 m di fronte al apparecchi contenenti mosche nelle fiale polistirolo, mettere a fuoco e lo zoom sul dispositivo, e impostare un timer a 3,0 secondi. <img alt="Figura 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> Dati rappresentativi Figura 2. Dal saggio di scansione delle larve con la mosca ectopicamente esprimere UAS FUS-WT e UAS-FUS R521C sotto il controllo di un driver motor neuron (OK371-GAL4). <pclass = &quot;jove_content&quot;&gt;

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Non abbiamo nulla da rivelare.

Drosophila comportamento è strettamente regolato da fattori genetici e ambientali. Noi, e altri, hanno già utilizzato i saggi descritti qui per raccogliere i dati per esaminare i geni legati a volare comportamenti e per la salute umana le malattie neurodegenerative modellati in Drosophila 5-19. Per il saggio strisciando, attenta selezione di 3 rd instar larve è un passo fondamentale. Se il trattamento con un farmaco, ci vorranno 10-15 minuti (o più a seconda del tipo e natura ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Saccarosio </td><td> Fisher Scientific </td><td> S5-500 </td></tr><tr><td> Agarosio </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6 oz Drosophila bottiglia </td><td> Genesee Scientific </td><td> 32-130 </td></tr><tr><td> Pennello (# 1) </td><td> Ted Pella, Inc. </td><td> 11859 </td></tr><tr><td> Vola componenti alimentari </td><td></td><td></td></tr><tr><td> Farina di mais </td><td> Fisher Scientific </td><td> NC9109741 </td></tr><tr><td> Agar </td><td> Genesee Scientific </td><td> 66-104 </td></tr><tr><td> Melassa </td><td> Fisher Scientific </td><td> NC9349176 </td></tr><tr><td> L&#39;acido propionico </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> Apice </td><td> 20-258 </td></tr><tr><td> Etanolo </td><td> PescatoreScientifico </td><td> BP2818-4 </td></tr><tr><td> Lievito </td><td> Genesee Scientific </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, il moscerino della frutta, è stato utilizzato per studiare i meccanismi molecolari di una vasta gamma di malattie umane come il cancro, malattie cardiovascolari e le varie malattie neurologiche 1. Abbiamo ottimizzato i test comportamentali semplici e robuste per la determinazione locomozione larvale, la capacità di arrampicata per adulti (test RING), e comportamenti di corteggiamento di Drosophila. Questi test comportamentali sono ampiamente applicabile per studiare il ruolo dei fattori genetici e ambientali sul comportamento fly. Capacità di scansione larvale può essere attendibilmente utilizzato per determinare i cambiamenti nelle fasi iniziali nelle abilità di larve striscianti Drosophila e anche per l&#39;esame effetto di farmaci o geni di malattie umane (in mosche transgeniche) sul loro locomozione. Il saggio scansione larvale diventa più applicabile se l&#39;espressione o la soppressione di un gene provoca mortalità negli stadi pupa o adulto, in quanto queste mosche non riescono a sopravvivere fino all&#39;età adulta, dove altrimenti potrebbero essere valutati. Questo di base unssay può essere utilizzato anche in combinazione con la luce o di stress per esaminare ulteriori risposte comportamentali in larve di Drosophila. Comportamento di corteggiamento è stato ampiamente usato per studiare le basi genetiche del comportamento sessuale, e può essere utilizzato anche per esaminare l&#39;attività e il coordinamento, così come l&#39;apprendimento e la memoria. Corteggiamento Drosophila comporta la sostituzione di diversi stimoli sensoriali tra visivo, uditivo, e segnali chemosensoriali tra maschi e femmine che portano a una complessa serie di comportamenti motori ben caratterizzati che culminano in copula di successo. Saggi di arrampicata per adulti tradizionali (geotassi negativi) sono noiosi, lavoro intensivo, e richiede tempo, con variazioni significative tra i diversi studi 2-4. La rapida iterativo geotassi negativi (RING) saggio 5 ha molti vantaggi rispetto a protocolli più ampiamente impiegati, fornendo un approccio velocità riproducibile, sensibile, e di alta quantificare locomotore adulti e negative geotassi behaviors. Nel saggio RING, genotipi diversi o trattamenti farmacologici possono essere testati contemporaneamente utilizzando gran numero di animali, con l&#39;high-throughput approccio rende più suscettibili per gli esperimenti di screening.

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Methods to Assay Drosophila Behavior

Drosophila melanogaster è un sistema genetico e comportamentale del modello trattabile che è stato utilizzato per comprendere le basi molecolari e cellulari di molti importanti processi biologici per oltre un secolo 1. Drosophila è stata ben sfruttata per acquisire conoscenze in base genetica del comportamento mosca.

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 ...

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Methods to Assay Drosophila Behavior

42.6K Views.  Louisiana State University Health Sciences Center. Drosophila melanogaster è un sistema genetico e comportamentale del modello trattabile che è stato utilizzato per comprendere le basi molecolari e cellulari di molti importanti processi biologici per oltre un secolo 1. Drosophila è stata ben sfruttata per acquisire conoscenze in base genetica del comportamento mosca.

Video: Methods to Assay Drosophila Behavior

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 ...

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Neuroscienze

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 ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Il comportamento larvale della drosofila in risposta alla stimolazione optogenetica dei neuroni olfattivi di rilevamento

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In Vivo Optical Calcium Imaging della plasticità sinaptica indotta dall'apprendimento in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Saggio meccanico di prevenzione dei conflitti per misurare il comportamento del dolore nei topi

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...