This work was supported by NIH P20GM103643 to I. Meng.

1. Preparazione di larve <ol><li> Per analizzare una larva desiderato per la locomozione o la preferenza di posizione, crescere le larve in condizioni standard per l&#39;età desiderata per saggiare 10 utilizzando fly cibo standard 11. </li><li> Fare un filtro a rete da stretching grado nylon serigrafica maglia su un imbuto. Fissare la rete al collo a imbuto con l&#39;elastico. Inserire l&#39;imbuto in un becher. </li><li> Per raccogliere larve per l&#39;analisi, paletta una spatola-pieno di cibo contenente foraggiamento larve dalla bottiglia coltura e lavare con acqua di rubinetto RT il filtro a rete, raccogliendo larve individuale direttamente dalla rete con un pennello 10. </li></ol> 2. Preparazione di Assay Tubes <ol><li> Per preparare per tappare le provette, bollire con attenzione un gel di agar 4% e versare in una capsula di Petri a una profondità di 1,5 cm. Ciò fornirà una spina per entrambi i lati del tubo quando animale è racchiuso. La soluzione al 4% è sufficientemente densa per evitare larve da penetrating i tappi. </li><li> Assicurarsi che i tubi siano puliti e chiari prima dell&#39;inserimento di larva. Se non lo sono, ciò può bloccare i movimenti vengano registrati. </li><li> Per garantire larve ha umidità sufficiente per il movimento, preparare una spruzzetta contenente una piccola quantità di acqua calda. Capovolgere la bottiglia, orientare attentamente presa verso un lavandino e spremere delicatamente per espellere l&#39;acqua dal tubo di prelievo. </li><li> Inserire la presa della bottiglia nella provetta. Comprimere il flacone per fornire vapore acqueo nella provetta fino a visualizzare un sottile strato di condensa sulla parete del tubo. Video 1 mostra una larva muove in un tubo con una quantità appropriata di umidità. </li><li> Per sigillare la larva nel tubo, rimuovere il gel di agar dello spessore di 1,5 cm dalla capsula di Petri e posizionare su una superficie mesh per permettere il flusso d&#39;aria sotto l&#39;agar in modo che una spina agar può essere inserito nel tubo. Premere un&#39;estremità del tubo dosaggio nel gel due volte per inserire due tappi di gel in una delle estremità. </li><li> Con un paintbcorrere, mettere una larva nel tubo di circa 1,5 pollici di profondità e sigillare il tubo premendo l&#39;estremità più vicina la larva nel gel di agar. La pressione risultante costringerà il secondo tappo di gel di agar dall&#39;estremità opposta e sigillare l&#39;animale all&#39;interno del tubo. </li><li> Porre le provette nello dispositivo MB5 Multi-Beam Drosophila Activity Monitor (DAM) e regolare la posizione delle valvole in modo che gli animali non possono andare oltre il campo dei sensori. </li><li> Per garantire che assay tubi non scivolare fuori infrarossi quadro di lettura del dispositivo durante il trasporto, tenere il tubo in posizione da un anello di fissaggio mastice intorno ogni tubo nel punto di contatto del dispositivo di registrazione. </li></ol> 3. Misurazione Attività <ol><li> L&#39;attività di registrazione in un incubatore per evitare letture errate che possono verificarsi a causa di ombre, luci fluorescenti o alle variazioni di temperatura in laboratorio. Vedere Figura 5 per una disposizione suggerita del sistema. </li><li> Impostare un incubatore a 20 ° C (vedi Figura 1). Per evitare falsi registrazioni dovuta a interferenze da fonti luminose ad incandescenza durante la registrazione, spegnere le luci fluorescenti e incubatori utilizzare una sorgente luminosa a LED separato durante la registrazione in incubatrice. Eseguire una prova senza animali per garantire che le condizioni di luce non aberrante innescare sensori. Non ci dovrebbero essere i dati di movimento registrati dopo questa prova. </li><li> Lasciare che le larve si adatti alle impostazioni di incubazione 20 ° C per 5 minuti prima dell&#39;inizio della dosaggio. </li><li> Per impostare il sistema DAM a intervalli di registrazione desiderata (ad esempio, 1 min), garantire software di registrazione di sistema DAM viene scaricato sul computer host 12 e aprire il file di sistema DAM prima di collegare il camera di registrazione all&#39;interfaccia psiu. </li><li> Per impostare la frequenza di registrazione desiderata in cui verranno salvati i dati, selezionare le preferenze e quindi fare clic sopra o sotto l&#39;opzione intervallo di lettura per selezionare differenti frazioni di tempo in cui i dativerrà memorizzato. Ad esempio, selezionare la lettura tempi di intervallo da 1 sec a 1 ora. </li><li> Per selezionare il parametro che varia per la registrazione dei dati, scegliere le preferenze e quindi selezionare le caselle corrispondenti sotto tipo di dati in uscita (conti, movimenti, posizioni e dimorare, vedi Tabella 1). Ciascun parametro fornisce un&#39;analisi unica dell&#39;attività larvale all&#39;interno del tubo. </li><li> Per iniziare la registrazione, collegare il monitor al Modulo di interfaccia di alimentazione (psiu) tramite cavo telefonico CAB6. Collegare il psiu ad una presa di corrente. Una luce verde indicherà connessione appropriata. </li><li> Collegare il psiu tramite un cavo Universal Serial Bus (USB) per un Macintosh o PC Windows per la registrazione dei dati. Aprire il programma DAM SystemMB1v6x sul computer per avviare automaticamente la registrazione dei dati. </li><li> Dopo il tempo di raccolta desiderato è completo, selezionare Esci e quindi chiudere ora sullo schermo l&#39;attività DAM; dati verrà salvato automaticamente sotto DAM dati di sistema. Prendete questo file di dati (ad esempio, monitorare 1) e trascinate<html> in una cartella separata. Ciò memorizzare i dati grezzi dal software DAM in modo che possano essere successivamente elaborati. </li></ol> 4. Preparazione dei dati per l&#39;elaborazione (DAM FileScan) <ol><li> Per elaborare i dati grezzi in un formato comprensibile aprire l&#39;applicazione DAM FileScan selezionare Seleziona cartella dei dati di ingresso. Quindi scegliere la cartella di dati e il file desiderato, e selezionare l&#39;opzione di scansione. Infine selezionare la lunghezza bin per il periodo di lettura desiderata. Ciò organizzare i dati grezzi in parametri impostati dall&#39;operatore ed il programma riporterà i dati raccolti in quel particolare intervallo. </li><li> Selezionare il tipo di dati di uscita per analizzare il movimento impostazione desiderata (conteggi del monitor, si muove, dimorano). Nel menu letture supplementari, somma selezionare in bin (questo è se la lunghezza bidone è maggiore dell&#39;intervallo di lettura sistema selezionato). </li><li> Nome del file in modo appropriato e salvare. Il file verrà memorizzato nella stessa posizione come la cartella dal punto 3.9. </li></ol>_title &quot;&gt; 5. Accesso ai dati per l&#39;analisi <ol><li> Al fine di raccogliere mosse media, elaborare i dati grezzi generati dal punto 4. Per generare una tabella in un foglio di calcolo, aprire il file che è stato salvato in precedenza (punto 4.3), e quando appare la finestra di importazione guidata testo, selezionare finitura per aprire i file di dati in formato foglio di calcolo. Nota: A seconda del tipo di dati analizzati, il foglio di calcolo verrà letto in modo diverso. In genere per misurare monitorare movimenti o conteggi, il periodo di tempo di studio sarà registrato numericamente, mentre ogni tubo lettura individuale riceverà una lettera designata. Quando si visualizzano i dati nel foglio di calcolo, le colonne KZ rappresentano gli slot per provette 1-16 rispettivamente. Le righe rappresentano i punti di dati raccolti. </li><li> Ad esempio, se sono stati raccolti muove per 20 min a intervalli di 1 minuto, la media dei 20 righe per calcolare un numero medio di mosse / minuto e altre misurazioni. </li></ol>

<ol><li>Tracey, W. D. Jr, Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((painless%2C+a+Drosophila.gene+essential+for+nociception%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">painless, a Drosophila.gene essential for nociception.</a> Cell. 113, 261-273 (2003).</li>
<li>Ainsley, J. A., Kim, M. J., Wegman, L. J., Pettus, J. M., Johnson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+mechanisms+controlling+the+timing+of+larval+developmental+and+behavioral+transitions+require+the+Drosophila.DEG%2FENaC+subunit%5BTitle%5D)+AND+%22Pickpocket1.+Dev.+Biol%22%5BJournal%5D)">Sensory mechanisms controlling the timing of larval developmental and behavioral transitions require the Drosophila.DEG/ENaC subunit.</a> Pickpocket1. Dev. Biol. 322 (1), 46-55 (2008).</li>
<li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+disease+models+in+Drosophila.+melanogaster.and+the+role+of+the+fly+in+therapeutic+drug+discovery%5BTitle%5D)+AND+%22Pharmacol.+Rev%22%5BJournal%5D)">Human disease models in Drosophila. melanogaster.and the role of the fly in therapeutic drug discovery.</a> Pharmacol. Rev. 63 (2), 411-436 (2011).</li>
<li>Cattaert, D., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blockade+of+the+central+generator+of+locomotor+rhythm+by+noncompetitive+NMDA+receptor+antagonists+in+Drosophila.larvae%5BTitle%5D)+AND+%22J.+Neurobiol%22%5BJournal%5D)">Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila.larvae.</a> J. Neurobiol. 48 (1), 58-73 (2001).</li>
<li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+analysis+of+larval+locomotion+in+Drosophila.+hordotonal+organ+mutants%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Dynamic analysis of larval locomotion in Drosophila. hordotonal organ mutants.</a> PNAS. 100 (26), 16053-16058 (2003).</li>
<li>Nichols, C. D., Bechnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+to+assay+Drosophila+behavior%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Methods to assay Drosophila behavior.</a> J. Vis. Exp. (61), (2012).</li>
<li>Godoy-Herrera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+and+genetics+of+digging+behavior+in+Drosophila.+arvae%5BTitle%5D)+AND+%22Heredity%22%5BJournal%5D)">The development and genetics of digging behavior in Drosophila. arvae.</a> Heredity. 56, 33-41 (1986).</li>
<li>Sinadinos, C., Cowan, C. M., Wyttenbach, A., Mudher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+throughput+assays+of+locomotor+dysfunction+in+Drosophila.+larvae%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Increased throughput assays of locomotor dysfunction in Drosophila. larvae.</a> Journal of Neuroscience Methods. 203 (2), 325-334 (2012).</li>
<li>Homyk, T., Sheppard, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+mutants+of+Drosophila+melanogaster.+I.+Isolation+and+mapping+of+mutations+which+decrease+flight+ability%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Behavioral mutants of Drosophila melanogaster. I. Isolation and mapping of mutations which decrease flight ability.</a> Genetics. 87 (1), 95-104 (1977).</li>
<li>Chattopadhyay, A., Gilstrap, A. V., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+and+global+methods+of+assessing+thermal+nociception+in+Drosophila.+larvae%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Local and global methods of assessing thermal nociception in Drosophila. larvae.</a> J. Vis. Exp. (63), e3837(2012).</li>
<li>Model Organisms I: yeast, Drosophila and C. elegans. Science Education Database. , JoVE. Cambridge, MA. Available from: 
 <a target="_blank" href="https://www.jove.com/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans">https://www.jove.com/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans</a> (2015).</li>
<li>Woods, J. K., Kowalski, S., Rogina, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+the+spontaneous+locomotor+activity+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Determination of the spontaneous locomotor activity in Drosophila melanogaster.</a> J. Vis. Exp. (86), e51449(2014).</li>
<li>Troncoso, B., Godoy-Herrera, R., Waldo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+larval+movement+patterns+in+Drosophila%29%5BTitle%5D)+AND+%22Heredity%22%5BJournal%5D)">The development of larval movement patterns in Drosophila).</a> Heredity. 58 (1), 321-329 (1987).</li>
<li>Helfrich, C., Engelmann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+the+locomotor+activity+in+Drosophila.+melanogaster.and+its+mutants+%E2%80%98sine+oculis%E2%80%99+and+%E2%80%98small+optic+lobes%5BTitle%5D)+AND+%22Physiological+Entomology%22%5BJournal%5D)">Circadian rhythm of the locomotor activity in Drosophila. melanogaster.and its mutants ‘sine oculis’ and ‘small optic lobes.</a> Physiological Entomology. 8 (3), 257-272 (1983).</li>
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The authors declare that they have no competing financial interests.

Attività di larve di Drosophila è influenzata da una varietà di fattori, tra genotipo 8, 13 anni e la temperatura ambiente 2. Sebbene potenti metodi videografici capaci di analisi molto dettagliate sono state sviluppate da parte di coloro che studiano locomozione 5, questo livello di dettaglio può essere superfluo per coloro che desiderano per determinare i parametri di base di attività. Il metodo qui descritto si avvale di un dispositivo che è disponibile in molti laborat...

The use of Drosophila as a genetic tool has transformed scientific knowledge of biological systems. Drosophila larvae have been used in a variety of studies including nociception1, development2 and as a model for the study of human disease genes3. Drosophila activity encompasses a range of behaviors that vary under different conditions including temperatures2, exposure to drugs4 and amongst different genotypes. Yet, despite the significant use of the larva as a model organism, a simple, standardized method to analyze larval activity has not been available. Presently, many larval locomotion studies employ sophisticated video analysis software5. While powerful, the complexity of such automated tools may discourage labs that are not already equipped to study locomotion from including analysis of informative activity parameters in their studies. In other current non-automated methods, such as the grid crawling analysis, motion is scored by a human observer, which introduces the possibility of subjectivity and limits throughput to one animal at a time6-7. A similar study used a 5-lane crawling assay, which measured the time it took larvae to travel a certain linear distance8. In such non-automated assays, displacement is measured but this does not account for non-linear travel between the start and end points. As discussed below, the method described here accounts for more of the actual larval movement, is objective, easy to operate, and offers robust throughput.
To easily study larval activity behavior without the compromise of accuracy, efficiency, or cost, this method employs the TriKinetics Drosophila Activity Monitor (DAM), a device often used to study adult activity. Using one device to study both adult and larval activity is cost-effective, and allows direct comparison of motion by animals at these two life stages. The system, featuring the manufacturer&#8217;s highest level of resolution, makes use of 17 infrared detection beams per assay tube, which record larval activity when sensor beams are broken within the 16 individual tubes. The system then automatically saves recorded information to a computer, making it available for manipulation with basic graphing software. The data obtained represents the beams that were broken by individual larvae (which can be converted into a rate), movement when a larva stays within a detector beam and the position of the animals within the assay chambers during a recording period (allowing one to calculate position preference). The system is efficient and relatively simple to operate, and brings highly reproducible basic activity analysis within the reach of any laboratory studying Drosophila larvae.
To demonstrate the power of this assay, data are presented that show its use to verify differences in activity resulting from varying ambient temperatures, as well as through the comparison of a mutant previously described as hypoactive (iav1)9 with a widely analyzed control (w1118).

<table><tbody><tr><td>Drosophila Activity Monitor, Multibeam, 16 tubes, including wires</td><td>TriKinetics Inc.&amp;nbsp;</td><td>MB5</td><td></td></tr><tr><td>Power Supply Interface for Activity Monitor&amp;nbsp;</td><td>TriKinetics Inc.&amp;nbsp;</td><td>PSIU24</td><td></td></tr><tr><td>Glass 80 x 5 mm tubes for Activity Monitor (100)</td><td>TriKinetics Inc.&amp;nbsp;</td><td>PGT 5x80</td><td></td></tr><tr><td>DAMsystemMB1v6x Data Acquisitions Software for Macintonsh OSX (Intel)</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>DAMFileScan 108x software for Macintosh</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>USB software (PSIUdrivers.zip).</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>DAMSystem Notes 308</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>Zeiss Stemi 2000C- Stereo Microscope</td><td>Spectra Services</td><td>SP-STEMI2000C-BS</td><td></td></tr><tr><td>Carbon Dioxide</td><td>Maine Oxy</td><td></td><td>anaesthesia</td></tr><tr><td>Fly Pad</td><td>Genesee</td><td>59-114</td><td>surface for sorting anaesthetized flies</td></tr><tr><td>Small paint brush&amp;nbsp;</td><td>Winsor &amp;amp; Newton</td><td>#2 ROUND</td><td>or similar, used for sorting anaesthetized flies</td></tr><tr><td>Silk Screen Printing Mesh (160)</td><td>msj-gallery.com</td><td>SM160W63-3YD</td><td>pore sized used in this protocol was ~ 0.1 mm</td></tr><tr><td>Tegosept</td><td>Genesee</td><td>20-258</td><td>preservative</td></tr><tr><td>Ethanol (190proof)</td><td>Pharmco</td><td>111000190</td><td>used to dissolve Tegosept</td></tr><tr><td>6 oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td></td></tr><tr><td>&quot;Flugs&quot; for Plastic Fly bottles</td><td>Genesee</td><td>49-100</td><td></td></tr><tr><td>Drosophila Vials, Wide (PS)</td><td>Genesee</td><td>32-117</td><td></td></tr><tr><td>Flugs for wide plastic vials</td><td>Genesee</td><td>49-101</td><td></td></tr><tr><td>Yellow Degerminated Corn Meal</td><td>Gold Medal</td><td></td><td></td></tr><tr><td>Drosophila agar</td><td>LabScientific</td><td>FLY 8020</td><td></td></tr><tr><td>Baker&#039;s Yeast - Red Star</td><td>King Arthur Flour</td><td>1270</td><td></td></tr><tr><td>Granulated Sugar - Extra Fine</td><td>Domino</td><td></td><td></td></tr></tbody></table>

measurement of larval activity in the drosophila activity monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

La Figura 1 mostra i risultati di uno studio di risposta di temperature di controllo larve al terzo stadio, w 1118, utilizzando il dispositivo di monitoraggio per rilevare le differenze di locomozione larvale a sette diverse temperature. Le larve sono state lavate e collocato nel dispositivo dell&#39;attività DAM come sopra descritto, e collocato in un incubatore alla temperatura desiderata. L&#39;apparecchio è stato poi consentito di acclimatarsi all&#39;ambiente per 5 minuti prima dell&#3...

Watch this Scientific Journal Video about Measurement of Larval Activity in the Drosophila Activity Monitor at JoVE.com

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Misura della larvali attività nel<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

measurement-of-larval-activity-in-the-drosophila-activity-monitor

Research

JoVE Journal

Biology

Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K Views.  University of New England. This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Video: Measurement of Larval Activity in the Drosophila Activity Monitor

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Saggio attività locomotoria per studiare ritmi circadiani del sonno e parametri in Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Ricerca

Neuroscienze

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Misurazione dei livelli di esercizio in Drosophila melanogaster utilizzando il sistema di quantificazione esercizio rotanti (Rich)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Genetica

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

Un metodo di calcolo per quantificare le attività circadiana volare

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Comportamento

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Misurazione quantitativa della risposta immunitaria e nel sonno<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Immunologia e infezioni

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Biologia

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 ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Un metodo a basso costo per l&#39;analisi sequestro-come attività e il movimento in<em&gt; Drosophila</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Una semplice tecnica per analizzare l'attività locomotoria nella Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

<meta charset="utf8"></head><body>Misura della tolleranza al calore ad alto rendimento in Drosophila utilizzando i sistemi DAM2

Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to assessing heat tolerance being to monitor the time to knockdown (TKD) after exposure to an elevated temperature. Classically, flies are housed in individual vials and placed inside a heated water bath. TKD is then monitored manually by researchers. While very well-established, there remain problems of subjectivity and consistent application of a tangible definition of cessation of all movement, including muscular spasms, when implementing these manual assays. We have developed a high-throughput method for automating heat tolerance assays using the TriKinetics Drosophila Activity Monitors (DAM2). To accompany the DAM2 system, we have written a program and created an easy-use executable to automatically read the last time of movement from the activity data generated. This script then writes to a .csv file the time to heat paralysis (TKD) for each fly. Our data show that this automated DAM2 method is consistent and reliable. Meanwhile, activity profiles created from the activity count data are of interest. These activity profiles can be compiled and have the potential to expand heat tolerance assays to include the relatively unstudied behavioral components of heat tolerance. This protocol will describe in detail how to use the DAM2 system and the HoTDAM! software to estimate heat tolerance in D. melanogaster.

<meta charset="utf8"></head><body>Saggi di tolleranza al calore con il sistema di monitoraggio dell'attività della Drosophila : una guida a un'applicazione eseguibile per l'analisi dei dati<

This report describes a method for measuring adult Drosophila melanogaster time to knockdown using a Drosophila Activity Monitor (DAM2) in response to an air conduction heat stressor within an incubator chamber. The DAM2 measures activity by recording individual fly movements as they cross an infrared beam. Data analysis is facilitated by a novel executable file created by the authors.

Saggi di tolleranza al calore con il sistema di monitoraggio dell'attività della Drosophila : una guida a un'applicazione eseguibile per l'analisi dei dati

Misura della larvali attività nel

Riepilogo

Esplora Altri Video

Saggio attività locomotoria per studiare ritmi circadiani del sonno e parametri in Drosophila

Misurazione dei livelli di esercizio in Drosophila melanogaster utilizzando il sistema di quantificazione esercizio rotanti (Rich)

Un metodo di calcolo per quantificare le attività circadiana volare

Misurazione quantitativa della risposta immunitaria e nel sonno

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

Determinazione del locomotore attività spontanea in

Un metodo a basso costo per l'analisi sequestro-come attività e il movimento in

Una semplice tecnica per analizzare l'attività locomotoria nella Drosophila

Saggi di tolleranza al calore con il sistema di monitoraggio dell'attività della Drosophila : una guida a un'applicazione eseguibile per l'analisi dei dati

Saggi di tolleranza al calore con il sistema di monitoraggio dell'attività della Drosophila : una guida a un'applicazione eseguibile per l'analisi dei dati