Wir danken Christian Landry für Hitze-Sonde Design, Daniel Babcock für die Entwicklung der Larven-Wärme-Sonde Assay, Sean Sweeney für die Annahme, die Hitze Platten-Assay, der Bloomington Drosophila Stock Center für Fliegenfischer Aktien und Galko Labor Mitglieder für die kritische Durchsicht des Manuskripts. Diese Arbeit wurde vom NIH R01 NS069828 zu MJG und einem NIH-MARC-U-STAR Training Grant (T34GM079088 an der University of Houston-Downtown Scholars Akademie) für den Zugang von Minderheiten, die wissenschaftliche Laufbahn (AVG) unterstützt.

<ol>
	<li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr. Biol. 19, 799-806 (2009).</li><li>Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=painless,+a+Drosophila+gene+essential+for+nociception.">painless, a Drosophila gene essential for nociception.</a> Cell. 113, 261-273 (2003).</li><li>Oswald, M., Rymarczyk, B., Chatters, A., Sweeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+thermosensitive+escape+behavior+in+Drosophila+larvae.">A novel thermosensitive escape behavior in Drosophila larvae.</a> Fly (Austin). 5, 17810 (2011).</li><li>Grueber, W. B., Jan, L. Y., Jan, Y. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Walters, E. T., Illich, P. A., Weeks, J. C., Lewin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+responses+of+larval+Manduca+sexta+and+their+sensitization+by+noxious+stimuli+in+the+laboratory+and+field.">Defensive responses of larval Manduca sexta and their sensitization by noxious stimuli in the laboratory and field.</a> J. Exp. Biol. 204, 457-469 (2001).</li><li>Woolf, C. J., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors--noxious+stimulus+detectors.">Nociceptors--noxious stimulus detectors.</a> Neuron. 55, 353-364 (2007).</li><li>Hwang, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr. Biol. 17, 2105-2116 (2007).</li><li>Xiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921-926 (2010).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Sweeney, N. T., Li, W., Gao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+single+neurons+in+vivo+reveals+specific+roles+of+flamingo+in+neuronal+morphogenesis.">Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis.</a> Dev. Biol. 247, 76-88 (2002).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12, 1195-1206 (1994).</li><li>Nitabach, M. N., Blau, J., Holmes, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+silencing+of+Drosophila+pacemaker+neurons+stops+the+free-running+circadian+clock.">Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock.</a> Cell. 109, 485-495 (2002).</li></ol>

Wir haben nichts zu offenbaren.

Die Assays können die hier beschriebenen verwendet werden, um qualitativ und quantitativ beurteilen Larven verschiedener Genotypen für das Ansprechen auf schädliche thermische Reize werden. Ein wesentliches Merkmal der Wärmesonde Test ist, dass der Reiz nur an einem einzigen Ort gegeben ist. Dies führt vermutlich zu Brennen nur einen kleinen Teil der Klasse IV-Neuronen diejenigen des Segments der Sonde in Kontakt gebracht und vielleicht die in unmittelbar benachbarten Segmenten 11. Aufgrund der lokalen Stimulation, ahmt die Hitze-Sonde Assay das gemeinsame sinnliche Erfahrung zum Nachweis eines schädlichen Reiz, die einer bestimmten Körperregion-wie eine Hand in Kontakt bringen einer heißen Herdplatte lokalisiert ist. Ein Nachteil der Wärmesonde Test ist, dass es einige Benutzer-zu-Benutzer-Variabilität, die wahrscheinlich drei Faktoren zurückzuführen ist: i) der Druck, mit dem der Benutzer gilt die Sonde in die Larve, ii) die genaue Position der Sonde auf der Larven zu den darunter liegenden nozizeptiven Neuronen, und iii) die genaue ANGle, bei der die Sonde die Oberfläche berührt der Larve. Wir zuvor berichteten über eine quantitative Strategie zur Kategorisierung Larven in nicht-Respondern, langsam Responder und schnelle Ansprechen auf ihren Wegziehlatenz auf eine bestimmte Temperatur 1 umfassen. Wir berichten hier über Larven Reaktionsfähigkeit auf noch höhere Temperaturen. Interessanterweise finden wir, dass es eine Decke, um Larven thermische nozizeptive Antworten und dass diese Obergrenze liegt zwischen 52 und 54 ° C. Dies deutet darauf hin, dass Larven besitzen keine transient receptor potential (TRP) befähigten Kanals Gating bei Temperaturen höher als 52 ° C Alternativ könnte es nahe, dass die Nervenzellen oder Muskeln verwendet zu initiieren oder durchführen die motorische Reaktion beschädigt werden, bevor sie überhaupt in aversiven Rückzug funktionieren kann. Wir berichten auch über eine andere Analyse der Amplitude des Entzugserfolges-entweder die Anzahl der Rollen als Indikator für die &quot;Robustheit&quot; der Antwort. Naiv, einwürde erwarten, dass diese Parameter würden mit zunehmender Temperatur oder Zeit der Stimulation zu erhöhen. Überraschenderweise finden wir, dass dies nicht der Fall ist. Larven für eine längere Zeit bei einer Temperatur am unteren Ende des schädlichen Bereich (42 ° C) stimuliert für mehr Walzen und mehr Zeit für die Walzen als Larven bei höheren Temperaturen (48-52 ° C) sondiert. Dies deutet darauf hin, dass innerhalb des schädlichen Temperaturfenster es in erster Linie ist die Dauer der Exposition, die die Amplitude der Reaktion bestimmt. Da Larven zu hoch schädlichen Temperaturen ausgesetzt sind (48-52 ° C) im Durchschnitt sehr schnell reagieren, sie haben nicht so viele Rollen als Larven ausgesetzt zu einer weniger schädlichen Temperatur für eine längere Zeitspanne aufweisen. Die Analyse der Reaktion Amplitude berichtet hier fügt eine weitere quantitative Dimension, entlang der verschiedenen Genotypen oder Umwelt-Manipulationen verglichen werden können. Ein Hauptmerkmal der Hitze Platten-Assay ist, dass es eine globale Exposition gegenüber dem schädlichen beinhaltetWärme. Als solches ist es eher dem Tier sitzt in einem Heiz-Kessel als Berühren einer heißen Herdplatte. Es ist zwar nicht klar, wann die Larven einen global schädlichen Reiz in freier Wildbahn erleben kann, im Labor die Verhaltensreaktionen auf diese globale Exposition sind komplexer als diejenigen auf lokaler Stimulation beobachtet. Eine Stärke des Wärme-Platten-Assay, auch von anderen 3 notiert haben, ist, dass es wenig User-to-User-Variabilität hat seit Berühren der Larve ist nicht Bestandteil des Protokolls. Der einzige wesentliche Abweichung scheint bei der Definition, wenn jedes Verhalten beginnt, und dies kann bei wiederholter Betrachtung / Vertrautheit minimiert werden kann. Ein interessanter Unterschied zwischen den Tests ist die Temperaturen, bei denen die aversive Verhaltensweisen beginnen. Dies sind deutlich niedriger in der Hitze Platten-Assay als mit der Hitze-Sonde. Das vorläufige Verhalten von Larven mit der Hitze-Sonde (Kopf und Schwanz raise) kontaktiert ausgestellt kann eine des Kopfes Thrash bei ~ 27 ° C in der Hitze Plattform beobachtet korrelieren seinE-Assay. Es ist möglich, dass diese Antwort &quot;Unbehagen&quot; über &quot;Schmerz&quot; widerspiegelt. Wir haben keine der Auspeitschung, Beschlagnahme korrelieren, und auch bei hohen Lähmungen beobachtet werden (bis zu 48 ° C) Temperaturen in den Hitze-Sonde Assay und es kann sein, dass eine kritische Masse an sensorischen Neuronen feuern aus mehr als einer Region des Körpers ist erforderlich, um auf diese Verhaltensweisen zu bringen. Unterhalb des unteren Endes des nozizeptiven Schwelle mit der Hitze-Sonde zeigt, dass globale Stimulation kann es sich um Addition von neuronalen Reaktionen, die nicht ausreichen, um das Verhalten mit den lokalen auslösen beobachtet - Interessanterweise werden die Beschlagnahme und die Lähmung Verhaltensweisen bei Temperaturen (37 ° C ~ 34) beobachtet Anwendung der Wärme-Sonde. Dass diese Temperaturen werden tatsächlich als schädlich für die Larven wahrgenommen wird durch die Beobachtung, dass Larven, die die Lähmung Verhalten beginnen und anschließend dürfen auf Fly Lebensmittel nicht wieder in den meisten Fällen nicht (5C) überleben unterstützt. Weitere unterstützende das Argument, dass die Wärme plgegessen Assay wird Auslesen nocizeptiven Reaktionen ist die Tatsache, dass die Blockierung der synaptischen Übertragung in bekannter nozizeptiven sensorischen Neuronen verlangsamt die Reaktion meisten der beobachteten Verhalten (Abbildung 6). Die Beobachtung, dass es keine Erhöhung der Latenz für die höhere Temperatur-Verhalten legt nahe, dass Auspeitschen anderen sensorischen Neuronen, die nicht exprimieren, MD-Gal4 für dieses Verhalten kann erforderlich sein. In der Summe umfassen beide Assays Belichten eines einzelnen Larve zu einer schädlichen thermischen Reiz des definierten Temperatur - die heiße Spitze eines kleinen Metall-Sonde in der lokalen Assay und das Eintauchen in einen Tropfen Wasser erwärmt sich schnell in der globalen Assay. Verhaltensreaktionen von Drosophila-Larven von unterschiedlichen genetischen Hintergrund und / oder ausgesetzt wechselnden Umgebungsbedingungen (zum Beispiel plus oder minus Gewebeschädigung), können studiert und quantifiziert werden unter Verwendung dieser Assays. Letztlich werden die Ergebnisse aus diesen Tests helfen uns besser zu verstehen, genetische Netzwerke Controlling nociception bei Drosophila und anderen verwandten Arten.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Thermische Sonde </td><td> Pro-Dev Technik </td><td> Auf Anfrage Custom-gebaut </td><td> Kontaktinformationen finden Sie auf Anfrage bereitgestellt werden </td></tr><tr><td> Trockenbad Incubator </td><td> Fisher Scientific </td><td> 11-718 </td><td> 1 feste Heizblock und 1 Heizblock mit 16mm Bohrungen </td></tr><tr><td> Leica DFC290 12v/400mA Farbkamera </td><td> Leica Microsystems </td><td> 12730080 </td><td> Eine äquivalente Kamera zu tun. </td></tr><tr><td> Leica MZ6 Mikroskop </td><td> Leica Microsystems </td><td> Teilenummer für MZ6 Zoom Körper (Optikträger) ist 10445614 </td><td></td></tr><tr><td> Schott Ace Modulamp Einheit </td><td> Schott North America, Inc. </td><td> A20500 </td><td></td></tr><tr><td> Schott Dual-Schwanenhals 23-Zoll-Lichtleitkabel </td><td> Schott North America, Inc. </td><td> Schott A08575 </td><td></td></tr><tr><td> Thermal Control Unit </td><td> TSCI corp. </td><td> Custom Built </td><td> Einzelheiten können auf Anfrage geliefert werden </td></tr><tr><td> Zeiss Stemi 2000-Mikroskop </td><td> Zeiss </td><td> NT55-605 </td><td> Eine äquivalente Mikroskoptun wird. </td></tr><tr><td> Zange </td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> 1mm Maschenweite </td><td> Genesee Scientific </td><td> 57-101 </td><td></td></tr><tr><td> Pinsel </td><td> Dick Blick Art Materials </td><td> 06762-1002 </td><td></td></tr><tr><td> UV-Vernetzer </td><td> Fisher Scientific </td><td> 1199289 </td><td></td></tr><tr><td> Färbetrog </td><td> Fisher Scientific </td><td> 08-816 </td><td></td></tr><tr><td> 10ml Becherglas </td><td> Fisher Scientific </td><td> 02-540C </td><td></td></tr><tr><td> Diethylether </td><td> Fisher Scientific </td><td> E138-500 </td><td></td></tr><tr><td> 35 x 10 mm Petrischale aus Polystyrol </td><td> Falke </td><td> 351008 </td><td> Wir haben noch nicht getestet Alternative Gerichte. </td></tr><tr><td> Glasobjektträger </td><td> Corning </td><td> 26003 </td><td></td></tr><tr><td> Thermoelement </td><td> Omega Engineering, Inc. </td><td> HH802U </td><td></td></tr><tr><td> Stück Vinyl </td><td> Office Depot </td><td> 480009 </td><td></td></tr><tr><td> Mikrozentrifugenröhrchen </td><td> Scientific DenvilleInc. </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

In diesem Artikel zeigen wir, Assays, um thermische Nozizeption in Drosophila-Larven zu studieren. Ein Test umfasst die räumlich begrenzten (lokalen) Stimulation von Nozizeptoren thermische 1,2, während die zweite besteht aus einem Großhandel (globale) Aktivierung der meisten oder alle diese Neuronen 3. Gemeinsam ermöglichen diese Techniken Visualisierung und Quantifizierung der Verhaltens-Funktionen von Drosophila nozizeptiven sensorischen Neuronen. Die Drosophila-Larve ist ein etabliertes Modellsystem, um die thermische Nozizeption, eine sensorische Reaktion auf potenziell schädlichen Temperaturen, die evolutionär über Artgrenzen 1,2 konserviert ist zu studieren. Die Vorteile von Drosophila für solche Studien sind die relative Einfachheit des Nervensystems und der Raffinesse der genetischen Techniken, die verwendet werden, um die molekulare Basis der zugrunde liegenden Biologie 4-6 in Drosophila sezieren kann, wie in allen Metazoen, die Response auf schädliche thermische Reize im Allgemeinen eine &quot;nozifensiven&quot; aversive Rückzug dem präsentierten Stimulus 7. Solche Impulse werden durch freie Nervenenden oder Nozizeptoren erkannt und die Amplitude des Organismus-Reaktion hängt von der Anzahl von Nozizeptoren Empfangen des schädlichen Reiz 8. Bei Drosophila sind es die Klasse IV dendritischen Verzweigungen sensorischen Neuronen, die schädliche thermische und mechanische Reize 9 erkennt zusätzlich zu ihrer Rolle als kürzlich entdeckten Photorezeptoren 10. Diese Neuronen, die sehr gut sind bei der Entwicklungsstufe studierte, über die Barriere epidermale Blatt verzweigen und Kontakte mit fast allen Epidermiszellen 11,12. Die einzelnes Axon jeder Klasse IV Neuron Projekte in den Bauchmark des zentralen Nervensystems 11, wo sie zu Neuronen zweiter Ordnung herstellen kann, dass Projekt an das Gehirn. Unter Ausgangsbedingungen, nozizeptiven sensorischen neurons wird erst ausgelöst, wenn ein relativ hoher Schwellenwert erreicht ist. Die beschriebenen Tests hier nehmen die Ermittler zu den Ausgangswerten Verhaltensreaktionen oder vermutlich die Sensibilisierung, die folgenden Gewebeschädigung erfolgt. Quantifizieren Jeder Test provoziert unterschiedliche, aber miteinander Bewegungsapparates Verhaltensreaktionen auf schädliche thermische Reize und ermöglicht die Forscher zu visualisieren und quantifizieren verschiedene Aspekte der thermischen Nozizeption in Drosophila-Larven. Die Tests können zu Larven der gewünschten Genotypen oder Larven unter verschiedenen Umweltbedingungen, die Nozizeption beeinflussen könnten erhoben angewendet werden. Da die thermische Nozizeption über Arten konserviert wird, werden die Erkenntnisse aus der genetischen Dissektion bei Drosophila gewonnenen wahrscheinlich informieren unser Verständnis der thermischen Nozizeption bei anderen Arten, einschließlich Wirbeltieren.

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae | Neuroscience | Video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In diesem Artikel zeigen wir, Assays, um thermische Nozizeption in studieren<em&gt; Drosophila</em&gt; Larven. Ein Test umfasst die räumlich begrenzten (lokalen) Stimulation der thermischen Nozizeptoren<sup&gt; 1,2</sup&gt;, Während die zweite besteht aus einem Großhandel (globale) Aktivierung der meisten oder alle diese Neuronen<sup&gt; 3</sup&gt;. Gemeinsam ermöglichen diese Techniken Visualisierung und Quantifizierung der Verhaltens-Funktionen<em&gt; Drosophila</em&gt; Nozizeptiven sensorischen Neuronen.

Lokale und globale Methoden zur Beurteilung der thermischen Nozizeption in<em&gt; Drosophila</em&gt; Larven

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

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Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Human Biology

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15.7K Aufrufe.  The University of Texas MD Anderson Cancer Center. In diesem Artikel zeigen wir, Assays, um thermische Nozizeption in studieren Drosophila Larven. Ein Test umfasst die räumlich begrenzten (lokalen) Stimulation der thermischen Nozizeptoren 1,2, Während die zweite besteht aus einem Großhandel (globale) Aktivierung der meisten oder alle diese Neuronen 3. Gemeinsam ermöglichen diese Techniken Visualisierung und Quantifizierung der Verhaltens-Funktionen Drosophila

Serie: Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Elektrophysiologische Messungen und Analyse von Nozizeption in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

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Methods to Assay Drosophila Behavior

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

Methods to Assay Drosophila Behavior

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

Methoden zum Test Drosophila Verhalten

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

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.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

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 ...

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.

Appetitive Assoziative olfaktorischen Lernens in<em&gt; Drosophila</em&gt; Larven

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.

Licht Vorlieben Assay zur angeborenen und Circadian geregelten Photobehavior in Studie<em&gt; Drosophila</em&gt; Larven

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 ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetische Störung der neuronalen Aktivität mit Laser Illumination in Semi-intakt<em&gt; Drosophila</em&gt; Larven in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Futter suchen Pfad-Länge-Protokoll für<em&gt; Drosophila melanogaster</em&gt; Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Neuartige Assay für kalte Nozizeption in<em&gt; Drosophila</em&gt; Larvae

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

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.

Ein Temperaturgradient Assay thermische Präferenzen der Drosophila Larven bestimmen

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

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Elektrophysiologische Methoden zur Messung des Photopigmentgehalts in Drosophila-Photorezeptoren

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...