Nous remercions Christian Landry pour réchauffage de la sonde de conception, Daniel Babcock pour le développement du dosage de la chaleur des larves de la sonde, Sean Sweeney pour suggérer le dosage de chaleur à plaques, la drosophile Bloomington Stock Center pour les stocks de mouches, et Galko membres du laboratoire pour la lecture critique du manuscrit. Ce travail a été soutenu par le NIH R01 NS069828 à MJG et une NIH MARC U-STAR de subventions de formation (T34GM079088 à l&#39;Université de Houston-Downtown Scholars Academy) pour l&#39;accès des minorités à des carrières de recherche (AVG).

<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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Moore, A. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hamlet,+a+binary+genetic+switch+between+single-+and+multiple-+dendrite+neuron+morphology.">hamlet, a binary genetic switch between single- and multiple- dendrite neuron morphology.</a> Science. 297, 1355-1358 (2002).</li><li>Gao, F. B., Brenman, J. E., Jan, L. Y., Jan, Y. 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>

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Les tests décrits ici peuvent être utilisés pour évaluer qualitativement et quantitativement les larves de différents génotypes de la réactivité aux stimuli nocifs thermiques. Une caractéristique principale de l&#39;essai sonde thermique, c&#39;est que le stimulus est donnée uniquement à un seul locus. Cela conduit sans doute à des tirs de seulement un petit sous-ensemble de la classe IV des neurones les plus dans le segment contactés par la sonde et peut-être ceux immédiatement les segments adjacents 11. En raison de la stimulation locale, le dosage sonde thermique imite l&#39;expérience sensoriel commun de détection d&#39;un stimulus nociceptif qui est localisée à un organe particulier la région comme une main en contact d&#39;un poêle chaud. Un inconvénient de l&#39;essai sonde thermique, c&#39;est qu&#39;il a une certaine variabilité d&#39;utilisateur à utilisateur qui peut probablement être attribuée à trois facteurs: i) la pression avec laquelle l&#39;utilisateur applique la sonde à la larve, ii) l&#39;emplacement précis de la sonde sur la chenille par rapport aux neurones sous-jacentes nociceptives, et iii) l&#39;précise angle auquel la sonde en contact avec la surface de la chenille. Nous avons précédemment fait état ​​d&#39;une stratégie quantitative des larves dans les catégories des non-répondeurs, répondeurs lents, et les intervenants rapides en fonction de leur temps de latence de retrait à une température donnée 1. Ici, nous rapportons sur la réactivité des larves à des températures encore plus élevées. Fait intéressant, nous trouvons qu&#39;il ya un plafond pour les larves thermiques réponses nociceptives et que ce plafond se situe entre 52 et 54 ° C. Cela peut indiquer que les larves ne possèdent pas un potentiel récepteur transitoire (TRP) canal capable de déclenchement à des températures supérieures à 52 ° C. Alternativement, il pourrait suggérer que les neurones ou les muscles utilisés pour initier ou mener à bien la réponse du moteur est endommagé avant même de pouvoir fonctionner en retrait aversif. Groupe présente également une analyse différente de l&#39;amplitude du retrait de réponse en utilisant-soit le nombre de rouleaux en tant qu&#39;indicateur de la &quot;robustesse&quot; de la réponse. Naïvement, ons&#39;attendrait à ce que ces paramètres augmentent avec la température augmente ou moment de la stimulation. Étonnamment, nous constatons que ce n&#39;est pas le cas. Les larves stimulé pendant une longue période à une température à l&#39;extrémité inférieure de la fourchette nocive (42 ° C) montrent plus de rouleaux et de passer plus de temps que les larves de roulement sondé à des températures élevées (48-52 ° C). Cela donne à penser que, dans la fenêtre de température nocive c&#39;est surtout la durée de l&#39;exposition qui détermine l&#39;amplitude de la réponse. Depuis que les larves exposées à des températures très nocives (48-52 ° C) de répondre, en moyenne, très rapidement, ils ne présentent pas sous forme de rouleaux plus grand nombre de larves exposées à une température moins nocive pour une plus longue période de temps. L&#39;analyse de l&#39;amplitude de réponse rapportés ici ajoute une autre dimension quantitative le long de laquelle différents génotypes ou des manipulations de l&#39;environnement peuvent être comparés. Une caractéristique principale de l&#39;essai de chaleur à plaques est qu&#39;il s&#39;agit d&#39;une exposition globale à l&#39;nocivesthermique. En tant que tel, il s&#39;apparente davantage à l&#39;animal assis dans un chaudron de chauffage que de toucher un poêle chaud. Même s&#39;il n&#39;est pas clair quand une larve peut vivre un stimulus nocif au niveau mondial à l&#39;état sauvage, dans le labo, les réponses comportementales à cette exposition globale sont plus complexes que ceux observés lors d&#39;une stimulation locale. Un des points forts de l&#39;essai de chaleur à plaques, a également noté par d&#39;autres 3, c&#39;est qu&#39;il a peu d&#39;utilisateur à utilisateur la variabilité, car tout contact de la larve n&#39;est pas une composante du protocole. L&#39;écart ne semble être importante dans la définition de chaque comportement quand commence et cela peut être minimisé avec visualisation répétée / familiarité. Une différence intéressante entre les tests est la température à laquelle les comportements d&#39;évitement commencer. Ce sont beaucoup plus faibles dans l&#39;essai de chaleur à plaques de la sonde thermique. Le comportement préliminaire présenté par les larves en contact avec la sonde thermique (tête et queue relance) peut être un corrélat de la tête de thrash observée à ~ 27 ° C dans le plat de la chaleure dosage. Il est possible que cette réponse reflète «malaise» plus que «la douleur». Nous n&#39;avons pas observé une corrélation entre la flagellation, la saisie, et la paralysie, même à doses élevées (jusqu&#39;à 48 ° C) la température dans le dosage de la sonde thermique et il se peut que d&#39;une masse critique de neurone sensoriel de tir de plus d&#39;une région du corps est nécessaires pour mettre sur ces comportements. Fait intéressant, les comportements de saisie et de la paralysie sont observées à des températures (~ 34 à 37 ° C) ci-dessous l&#39;extrémité inférieure du seuil nociceptif observé avec la sonde thermique indiquant que la stimulation globale peut impliquer sommation des réponses neuronales qui sont insuffisantes pour déclencher un comportement avec les autorités locales application de la sonde thermique. Que ces températures sont effectivement perçu comme nocif pour les larves est soutenue par l&#39;observation que les larves qui commencent le comportement paralysie et sont ensuite autorisés à récupérer la nourriture volée ne pas survivre à la plupart des cas (figure 5C). En outre soutenir l&#39;argument selon lequel la chaleur pldosage est mangé lire les réponses nociceptives est le fait que le blocage de la transmission synaptique dans les neurones sensoriels nociceptifs connus augmente la latence de la plupart des comportements observés (Figure 6). L&#39;observation selon laquelle il n&#39;y a pas augmentation de la latence pour le comportement à plus haute température à fouetter suggère que d&#39;autres neurones sensoriels qui n&#39;expriment pas md-Gal4 peut être nécessaire pour ce comportement. En somme, les deux tests implique d&#39;exposer une larve individu à un stimulus nociceptif thermique de la température définie - la pointe chaude d&#39;une petite sonde métallique dans le dosage locale et l&#39;immersion dans une goutte de rapidement chauffer l&#39;eau dans le dosage global. Les réponses comportementales de larves de drosophile de divers horizons génétiques et / ou exposés à des conditions environnementales variables (par exemple plus ou moins des dommages aux tissus), peuvent être étudiés et quantifiés à l&#39;aide de ces tests. En fin de compte, les résultats de ces essais nous aideront à mieux comprendre les réseaux génétiques qui contrôlent la CNPiception chez la drosophile et d&#39;autres espèces apparentées.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Sonde Thermique </td><td> Pro-Dev génie </td><td> Custom-construits sur demande </td><td> Information de contact peuvent être fournis sur demande </td></tr><tr><td> Bain à sec Incubateur </td><td> Fisher Scientific </td><td> 11-718 </td><td> 1 bloc de chauffage solide et 1 bloc de chauffage avec des puits 16mm </td></tr><tr><td> Leica DFC290 12v/400mA Caméra couleur </td><td> Leica Microsystems </td><td> 12730080 </td><td> Toute l&#39;équivalent appareil photo fera l&#39;affaire. </td></tr><tr><td> Leica MZ6 microscope </td><td> Leica Microsystems </td><td> Numéro de la pièce pour MZ6 corps de zoom (optique porteuse) est 10445614 </td><td></td></tr><tr><td> Schott Ace Modulamp Unité </td><td> Schott North America, Inc </td><td> A20500 </td><td></td></tr><tr><td> Schott col de cygne double 23 pouces guide de lumière à fibre optique </td><td> Schott North America, Inc </td><td> Schott A08575 </td><td></td></tr><tr><td> Unité de contrôle thermique </td><td> TSCI corp. </td><td> Custom Built </td><td> Les détails peuvent être fournis sur demande </td></tr><tr><td> Zeiss Stemi 2000 microscope </td><td> Zeiss </td><td> NT55-605 </td><td> Toute l&#39;équivalent microscopefera l&#39;affaire. </td></tr><tr><td> Forceps </td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> 1mm mailles </td><td> Genesee scientifique </td><td> 57-101 </td><td></td></tr><tr><td> Pinceau </td><td> Dick Blick Art Materials </td><td> 06762-1002 </td><td></td></tr><tr><td> Agent de réticulation UV </td><td> Fisher Scientific </td><td> 1199289 </td><td></td></tr><tr><td> Jarres </td><td> Fisher Scientific </td><td> 08-816 </td><td></td></tr><tr><td> Bécher 10ml </td><td> Fisher Scientific </td><td> 02-540C </td><td></td></tr><tr><td> L&#39;éther diéthylique </td><td> Fisher Scientific </td><td> E138-500 </td><td></td></tr><tr><td> 35 x 10 mm boîte de Pétri en polystyrène </td><td> Faucon </td><td> 351008 </td><td> Nous n&#39;avons pas testé plats de substitution. </td></tr><tr><td> Lame de microscope </td><td> Corning </td><td> 26003 </td><td></td></tr><tr><td> Thermocouple </td><td> Omega Engineering, Inc </td><td> HH802U </td><td></td></tr><tr><td> Morceau de vinyle </td><td> Office Depot </td><td> 480009 </td><td></td></tr><tr><td> Microtube </td><td> Denville scientifiqueInc </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

Dans cet article, nous démontrons essais pour étudier la nociception thermique larves de drosophile. Un test implique une stimulation spatialement restreint (local) de 1,2 nocicepteurs thermiques tandis que le second implique un gros (global) d&#39;activation de la plupart ou tous les neurones de ces 3. Ensemble, ces techniques permettent de visualiser et de quantifier les fonctions comportementales de Drosophila neurones sensoriels nociceptifs. La larve drosophile est un système modèle établi pour étudier la nociception thermique, une réponse sensorielle à des températures potentiellement dangereux qui est évolutif conservés entre espèces 1,2. Les avantages de la drosophile pour de telles études sont la relative simplicité de son système nerveux et de la sophistication des techniques génétiques qui peuvent être utilisés pour disséquer la base moléculaire de la biologie sous-jacente 4-6 dans la drosophile, comme dans tous les métazoaires, les responsabe à des stimuli thermiques nocifs implique généralement une &quot;nocifensive« retrait aversion pour le stimulus présenté 7. Ces stimuli sont détectés par les terminaisons nerveuses libres ou nocicepteurs et l&#39;amplitude de la réponse organismique dépend du nombre de nocicepteurs recevant le stimulus nociceptif 8. Chez la drosophile, ce sont les neurones de classe IV arborisation dendritique sensoriels qui détectent des stimuli nocifs thermiques et mécaniques 9 en plus de leur rôle récemment découvert que les photorécepteurs 10. Ces neurones, qui ont été très bien étudiés au niveau de développement, s&#39;arborisent sur ​​la feuille de la barrière épidermique et établir des contacts avec presque toutes les cellules épidermiques 11,12. Le seul axone de chaque neurone projets de classe IV dans la moelle épinière ventrale du système nerveux central 11 où ils peuvent se connecter à neurones de second ordre que le projet vers le cerveau. Dans des conditions de base, neuro nociceptif sensorielns ne se déclenche pas jusqu&#39;à un seuil relativement élevé est atteint. Les tests décrits ici permettent au chercheur de quantifier les réponses comportementales de base ou, vraisemblablement, la sensibilisation qui en découle des dommages aux tissus qui suit. Chaque test provoque distincts mais connexes locomotrices réponses comportementales aux stimuli nocifs thermiques et permet au chercheur de visualiser et de quantifier les différents aspects de la nociception thermique larves de drosophile. Les tests peuvent être appliqués pour les larves de génotypes désirés ou larves élevées dans différentes conditions environnementales qui pourraient avoir une incidence nociception. Depuis la nociception thermique est conservé à travers les espèces, les résultats glanés dans la dissection génétique chez la drosophile sera probablement notre compréhension de la nociception thermique dans d&#39;autres espèces, y compris les vertébrés.

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

Dans cet article, nous démontrons essais pour étudier la nociception thermique<em&gt; Drosophila</em&gt; Larves. Un test implique une stimulation spatialement restreint (local) des nocicepteurs thermiques<sup&gt; 1,2</sup&gt; Tandis que le second implique un gros (global) d&#39;activation de la plupart ou tous les neurones de ces<sup&gt; 3</sup&gt;. Ensemble, ces techniques permettent de visualiser et de quantifier les fonctions comportementales de<em&gt; Drosophila</em&gt; Nociceptives des neurones sensoriels.

Méthodes locales et globales de l&#39;évaluation de nociception thermique dans<em&gt; Drosophila</em&gt; Les larves

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

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15.7K vues.  The University of Texas MD Anderson Cancer Center. Dans cet article, nous démontrons essais pour étudier la nociception thermique Drosophila Larves. Un test implique une stimulation spatialement restreint (local) des nocicepteurs thermiques 1,2 Tandis que le second implique un gros (global) d'activation de la plupart ou tous les neurones de ces 3. Ensemble, ces techniques permettent de visualiser et de quantifier les fonctions comportementales de Drosophila

Vidéo: 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.

Mesures électrophysiologiques et d&#39;analyse de la nociception chez les nourrissons humains

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.

Méthodes de dosage à Drosophila Comportement

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.

Visualisation des propriocepteurs dans<em&gt; Drosophila</emLes larves&gt; et pupes

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.

Appétitive apprentissage olfactif associatif en<em&gt; Drosophila</em&gt; Les larves

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.

Essai de préférence la lumière pour étudier innée et circadien Photobehavior Réglementé<em&gt; Drosophila</em&gt; Les larves

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.

Optogenetic Perturbation de l&#39;activité neuronale avec illumination laser à semi-intactes<em&gt; Drosophila</em&gt; Les larves 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.

Foraging Protocole Chemin de longueur pour<em&gt; Drosophila melanogaster</em&gt; larves

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.

Nouveau test pour froid dans nociception<em&gt; drosophile</em&gt; larves

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.

Un dosage de Gradient de température pour déterminer les préférences thermiques de larves de drosophile

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;

Méthodes électrophysiologiques pour mesurer les niveaux de photopigment dans les photorécepteurs de la drosophile

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