Ringraziamo Christian Landry per la progettazione di calore sonda, Daniel Babcock per lo sviluppo del larve test sonda termica, Sean Sweeney per aver suggerito il saggio di calore a piastre, il Bloomington Drosophila Stock Center per gli stock finestre, e Galko membri di laboratorio per la lettura critica del manoscritto. Questo lavoro è stato supportato dal NIH R01 NS069828 a MJG e NIH MARC U-STAR Training Grant (T34GM079088 alla University of Houston-Downtown Scholars Academy) per l&#39;accesso a carriere di ricerca minoranza (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>

Non abbiamo nulla da rivelare.

I saggi descritti qui possono essere utilizzate per valutare qualitativamente e quantitativamente larve di genotipi differenti reattività a stimoli termici nocivi. Una caratteristica principale della sonda per saggi di calore è che lo stimolo viene dato solo un singolo locus. Questo porta alla cottura di presumibilmente solo un piccolo sottoinsieme di classe IV neuroni quelli del segmento contattato dalla sonda e forse quelli immediatamente adiacenti segmenti 11. A causa della stimolazione locale, la sonda per saggi di calore imita l&#39;esperienza comune sensoriale di rilevare uno stimolo nocivo che è localizzato in una particolare regione del corpo, ad esempio una mano a contatto una stufa calda. Uno svantaggio della sonda per saggi di calore è che ha qualche utente a utente variabilità che può essere attribuita a tre fattori: i) la pressione con cui l&#39;utente applica la sonda per la larva, ii) la posizione precisa della sonda la larva rispetto ai neuroni sottostanti nocicettivi, e iii) il preciso angLe in cui la sonda contatto con la superficie della larva. Abbiamo precedentemente riportato una strategia quantitativa di larve categorizzare in non responder, le risposte lenti e responder veloci in base alla loro latenza ritiro ad una data temperatura 1. Qui riportiamo la risposta delle larve a temperature ancora più alte. È interessante notare, troviamo che c&#39;è un tetto massimo per le risposte larvali termici nocicettivi e che tale massimale si trova tra 52 e 54 ° C. Questo può indicare che le larve non possiedono un potenziale transitoria recettore (TRP) canali in grado di gating a temperature superiori a 52 ° C. In alternativa, si potrebbe suggerire che i neuroni o dei muscoli utilizzati per avviare o effettuare la risposta del motore danneggiato prima di poter funzionare anche in ritiro avversione. Si riportano anche una diversa analisi della ampiezza della risposta-ritiro utilizzando il numero di rulli come indicatore della &quot;robustezza&quot; della risposta. Ingenuamente, unosi aspetta che questi parametri aumenta con l&#39;aumentare della temperatura o il tempo di stimolazione. Sorprendentemente, abbiamo trovato che questo non è il caso. Le larve stimolato per un tempo più lungo a una temperatura alla fine della gamma bassa nocive (42 ° C) mostrano più rotoli e più tempo trascorso rollio che larve sondati a temperature più elevate (48-52 ° C). Questo suggerisce che all&#39;interno della finestra di temperatura nocivo è soprattutto la durata di esposizione che determina l&#39;ampiezza della risposta. Poiché larve esposto a temperature estremamente nocivi (48-52 ° C) rispondono mediamente molto rapidamente, essi non presentano più rollini larve esposto ad una temperatura inferiore nocivo per un periodo di tempo più lungo. L&#39;analisi dell&#39;ampiezza della risposta qui riportati aggiunge un&#39;altra dimensione quantitativa lungo le quali genotipi differenti o manipolazioni ambientali possono essere confrontati. Una caratteristica principale del saggio piastra calore è che coinvolge una esposizione globale al nocivicalore. Come tale, essa è più simile a l&#39;animale seduto in un calderone di riscaldamento che toccare una stufa calda. Anche se non è chiaro quando una larva potrebbe verificare uno stimolo nocivo a livello globale in natura, in laboratorio le risposte comportamentali a questa esposizione globale sono più complessi di quelli osservati dopo stimolazione locale. Una forza del saggio a piastre, anche rilevato da altri 3, è che ha poco da utente a utente variabilità poiché tocca la larva non è un componente del protocollo. L&#39;unica variazione sostanziale sembra essere nel definire quando inizia ogni comportamento e questo può essere minimizzato con la visione ripetuta / familiarità. Una differenza interessante tra i saggi sono le temperature a cui i comportamenti avversative avviano. Questi sono molto inferiore nel test di calore a piastre che con la sonda di calore. Il comportamento preliminare esibita da larve a contatto con la sonda di calore (testa e coda rilancio) può essere correlato della testa thrash osservato a ~ 27 ° C in calore la piattaformae dosaggio. E &#39;possibile che questa risposta riflette &quot;fastidio&quot; più di &quot;dolore&quot;. Non abbiamo osservato una correlazione di frustate, convulsioni, paralisi e anche ad elevate (fino a 48 ° C) le temperature nel saggio della sonda di calore e può essere che una massa critica di neuroni sensoriali sparare da più di una regione del corpo è necessari per portare a questi comportamenti. È interessante notare, i comportamenti epilettiche e paralisi sono osservati a temperature (~ 34 - 37 ° C) sotto l&#39;estremità inferiore della soglia nocicettiva osservata con la sonda termica che indica che la stimolazione globale può comportare somma delle risposte neuronali che sono insufficienti per innescare con comportamento locale applicazione della sonda calore. Tali temperature sono effettivamente percepito come nocivi per le larve è supportata dall&#39;osservazione che le larve che iniziano il comportamento paralisi e sono successivamente lasciato recuperare il cibo fly non nella maggioranza dei casi sopravvivere (Figura 5C). Ulteriori sostenere la tesi secondo cui il calore plsaggio viene mangiato lettura risposte nocicettive è il fatto che bloccano la trasmissione sinaptica noti in neuroni sensoriali nocicettivi aumenta la latenza della maggior parte dei comportamenti osservati (Figura 6). L&#39;osservazione che non vi sia aumento della latenza per l&#39;alta temperatura comportamento frustate suggerisce che altri neuroni sensoriali che non esprimono md-Gal4 può essere richiesto per questo comportamento. In sintesi, entrambi i saggi comportano l&#39;esposizione di un individuo larva di uno stimolo nocivo termico di temperatura definito - la punta calda di una sonda metallica nel saggio locale e immersione in una goccia di rapido riscaldamento dell&#39;acqua nel saggio globale. Risposte comportamentali di Drosophila larve di vario background genetici e / o esposti a diverse condizioni ambientali (ad esempio, più o meno danni ai tessuti), possono essere studiate e quantificate con questi test. In definitiva, i risultati ottenuti in questi test ci aiuterà a capire meglio le reti genetiche che controllano nociception in Drosophila e di altre specie affini.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Sonda termica </td><td> Pro-Dev Ingegneria </td><td> Custom-costruita su richiesta </td><td> Informazioni di contatto possono essere fornite su richiesta </td></tr><tr><td> Dry Bath Incubator </td><td> Fisher Scientific </td><td> 11-718 </td><td> 1 blocco riscaldante solida e 1 blocco riscaldante con pozzi 16mm </td></tr><tr><td> Leica DFC290 12v/400mA Telecamera a colori </td><td> Leica Microsystems </td><td> 12730080 </td><td> Qualsiasi equivalente fotocamera farà. </td></tr><tr><td> Leica MZ6 microscopio </td><td> Leica Microsystems </td><td> Codice per MZ6 corpo zoom (corpo ottico) è 10445614 </td><td></td></tr><tr><td> Schott Ace Modulamp Unità </td><td> Schott North America, Inc. </td><td> A20500 </td><td></td></tr><tr><td> Schott doppio collo di cigno 23 pollici Light Guide fibra ottica </td><td> Schott North America, Inc. </td><td> Schott A08575 </td><td></td></tr><tr><td> Unità di controllo termico </td><td> TSCI corp. </td><td> Custom Built </td><td> I dettagli possono essere forniti su richiesta </td></tr><tr><td> Zeiss Stemi 2000 microscopio </td><td> Zeiss </td><td> NT55-605 </td><td> Qualsiasi equivalente microscopiofarà. </td></tr><tr><td> Forcipe </td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> 1 millimetro rete </td><td> Genesee Scientific </td><td> 57-101 </td><td></td></tr><tr><td> Pennello </td><td> Dick Blick Art Materials </td><td> 06762-1002 </td><td></td></tr><tr><td> UV reticolante </td><td> Fisher Scientific </td><td> 1199289 </td><td></td></tr><tr><td> Coplin </td><td> Fisher Scientific </td><td> 08-816 </td><td></td></tr><tr><td> 10ml beaker </td><td> Fisher Scientific </td><td> 02-540C </td><td></td></tr><tr><td> Etere etilico </td><td> Fisher Scientific </td><td> E138-500 </td><td></td></tr><tr><td> 35 X 10 mm Dish polistirolo Petri </td><td> Falco </td><td> 351008 </td><td> Non abbiamo testato piatti alternativi. </td></tr><tr><td> Glass vetrino da microscopio </td><td> Corning </td><td> 26003 </td><td></td></tr><tr><td> Termocoppia </td><td> Omega Engineering, Inc. </td><td> HH802U </td><td></td></tr><tr><td> Pezzo di vinile </td><td> Office Depot </td><td> 480009 </td><td></td></tr><tr><td> Microcentrifuga tubo </td><td> Denville scientificoInc. </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

In questo articolo dimostriamo saggi per studiare nocicezione termica in larve di Drosophila. Un test comporta la stimolazione spazialmente limitata (locale) di energia termica 1,2 nocicettori, mentre il secondo comporta una wholesale (globale) l&#39;attivazione della maggior parte o tutti questi neuroni 3. Insieme, queste tecniche consentono la visualizzazione e la quantificazione delle funzioni comportamentali di Drosophila neuroni sensoriali nocicettivi. La larva di Drosophila è un sistema modello per studiare istituito nocicezione termica, una risposta sensoriale, a temperature potenzialmente dannosi che è evolutivamente conservata tra le specie 1,2. I vantaggi di Drosophila per tali studi sono la relativa semplicità del suo sistema nervoso e la sofisticazione delle tecniche genetiche che possono essere utilizzati per sezionare la base molecolare della biologia sottostante 4-6 in Drosophila, come in tutti metazoi, i response nocivi a stimoli termici comporta generalmente un ritiro &quot;nocifensive&quot; avversione allo stimolo presentato 7. Tali stimoli vengono rilevati attraverso terminazioni nervose libere o nocicettori e l&#39;ampiezza della risposta organica dipende dal numero di nocicettori ricevono il stimolo nocivo 8. In Drosophila, è la classe IV arborizzazione dendritica neuroni sensoriali che rilevano nocivi stimoli termici e meccanici 9 in aggiunta al loro ruolo recentemente scoperto come fotorecettori 10. Questi neuroni, che sono stati studiati molto bene a livello di sviluppo, arborize sopra il foglio di barriera epidermica e di stabilire contatti con quasi tutte le cellule epidermiche 11,12. Il singolo assone di ogni classe IV progetti neuroni nel midollo nervoso ventrale del sistema nervoso centrale, 11 in cui possono connettersi al secondo ordine i neuroni che proiettano al cervello. In condizioni basali, neuro sensoriale nocicettivans non scatta finché una soglia relativamente elevata viene raggiunta. I saggi qui descritte consentono al ricercatore di quantificare le risposte comportamentali di base o, presumibilmente, la sensibilizzazione che ne deriva danni tessuto nel modo seguente. Ciascun test provoca risposte locomotorie distinti ma correlati comportamentali nocivi stimoli termici e consente al ricercatore di visualizzare e quantificare i vari aspetti della nocicezione termica in larve di Drosophila. I saggi possono essere applicati a larve di genotipi desiderati o di larve sollevata in diverse condizioni ambientali che potrebbero influenzare nocicezione. Dal momento che nocicezione termica è conservata tra le specie, i risultati raccolti da dissezione genetica in Drosophila è probabile che informerà la nostra comprensione di nocicezione termica in altre specie, tra vertebrati.

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 questo articolo dimostriamo saggi per studiare nocicezione termica<em&gt; Drosophila</em&gt; Larve. Un test comporta la stimolazione spazialmente limitata (locale) dei nocicettori termici<sup&gt; 1,2</sup&gt; Mentre il secondo comporta una wholesale (globale) l&#39;attivazione della maggior parte o tutti questi neuroni<sup&gt; 3</sup&gt;. Insieme, queste tecniche consentono la visualizzazione e la quantificazione delle funzioni comportamentali di<em&gt; Drosophila</em&gt; Neuroni sensoriali nocicettivi.

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

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

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Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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15.7K Views.  The University of Texas MD Anderson Cancer Center. In questo articolo dimostriamo saggi per studiare nocicezione termica Drosophila Larve. Un test comporta la stimolazione spazialmente limitata (locale) dei nocicettori termici 1,2 Mentre il secondo comporta una wholesale (globale) l'attivazione della maggior parte o tutti questi neuroni 3. Insieme, queste tecniche consentono la visualizzazione e la quantificazione delle funzioni comportamentali di Drosophila

Video: 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.

Le misurazioni elettrofisiologiche e Analisi di nocicezione nei neonati umani

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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Neuroscienze

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.

Metodi per test Drosophila Comportamento

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

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

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

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

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

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

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

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

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

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

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

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 Perturbazione di attività neurale con illuminazione laser in semi-intatte<em&gt; Drosophila</em&gt; Larve 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.

Foraggiamento protocollo percorso di lunghezza per<em&gt; Drosophila melanogaster</em&gt; Le larve

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.

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

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

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

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

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

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

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

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

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;

Metodi elettrofisiologici per misurare i livelli di fotopigmento nei fotorecettori di Drosophila

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