Name: a temperature gradient assay to determine thermal preferences of drosophila larvae
Uploaded: 2018-06-25T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

C.M. (DC007864, DC016278) NIDCD (EY008117, EY010852), NEI fon tarafından desteklenmektedir ve NIAID (1DP1AI124453).

<ol>
	<li>Fowler, M. A., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRP+channels+and+animal+behavior.">Drosophila TRP channels and animal behavior.</a> Life Sci. 92, 394-403 (2013).</li><li>Palkar, R., Lippoldt, E. K., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+thermosensation+in+mammals.">The molecular and cellular basis of thermosensation in mammals.</a> Curr Opin Neurobiol. 34, 14-19 (2015).</li><li>Vriens, J., Nilius, B., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+thermosensation+in+mammals.">Peripheral thermosensation in mammals.</a> Nat Rev Neurosci. 15 (9), 573-589 (2014).</li><li>Rosenzweig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li><li>Kwon, Y., Shim, H. S., Wang, X., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+thermotactic+behavior+via+coupling+of+a+TRP+channel+to+a+phospholipase+C+signaling+cascade.">Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade.</a> Nat Neurosci. 11, 871-873 (2008).</li><li>Kwon, Y., Shen, W. L., Shim, H. S., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+thermotactic+discrimination+between+the+optimal+and+slightly+cooler+temperatures+via+a+TRPV+channel+in+chordotonal+neurons.">Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons.</a> J Neurosci. 30 (31), 10465-10471 (2010).</li><li>Shen, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+rhodopsin+in+temperature+discrimination+in+Drosophila.">Function of rhodopsin in temperature discrimination in Drosophila.</a> Science. 331 (6022), 1333-1336 (2011).</li><li>Sokabe, T., Chen, H. S., Luo, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+switch+in+thermal+preference+in+Drosophila+larvae+depends+on+multiple+rhodopsins.">A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins.</a> Cell Rep. 17, 336-344 (2016).</li><li>Ni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ionotropic+Receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila.">The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila.</a> Elife. 5, 13254 (2016).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigational+decision+making+in+Drosophila+thermotaxis.">Navigational decision making in Drosophila thermotaxis.</a> J Neurosci. 30 (12), 4261-4272 (2010).</li><li>Ashburner, M., Golic, K. G., Hawley, R. 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> Drosophila: a laboratory handbook. , (2005).</li></ol>

Yazarlar ifşa gerek yok.

Bu protokolü başarılı olmasını sağlamak için larva deneyler yapmak için yeterli sayıda elde etmek için ilgili adımları uygulamak önemlidir. Bu Maya tüpler için 2-3 yumurta atarken geliştirmek d Yapıştır içeren sinekli ön besleme içerir. Şişeleri su şişeleri içeren bir tepsiye yerleştirilir ve gıda nem korur ve etkili tarafından larvaları besleme iken normal koyu döngüleri maruz izin teşvik şeffaf plastik bir torbaya alınmış gerekir. Ancak, Maya Yapıştır sinekler tuzak haline çok...

Thermotaxis hareket eden hayvanlar tarafından en uygun koşulları1,2,3bir ortam seçmek için istihdam edilmektedir. Eğer iklim aşırı sıcak veya soğuk, bu davranış yaşam için hayati önem taşımaktadır. Buna ek olarak, birçok hayvan çok küçük rahat aralığında sıcaklık farklılıkları duyarlıdır ve çevresi ile ideal bir sıcaklık ararlar. Bu onların vücut ısısı çevre ile equilibrate meyve sinekleri gibi poikilothermic organizmalar için belirli önem taşıyor. Larva thermotaxis izlemek için deneyleri ve moleküler sensörler Drosophila geçici potansiyel reseptör (TRP) kanalları4,5,6gibi rolleri açıklığa kavuşturulması enstrümantal oldunuz. rhodopsins7,8ve ionotropic reseptör reseptörleri (IRS)9, hangi sıcaklık hassasiyeti ile bu hayvanların farklı sıcaklık aralıklarında bağışlamak.
Bir iki yönlü seçim test termal larvaları6,7tercihlerinde çalışmaya bir yaklaşım sağlar. Tahlil iki farklı sıcaklık bölgeleri kurulması üzerine kuruludur ve hayvanlar üzerinde başka bir tarafı seçmek izin verir. Çift yönlü seçenek test sonuçlarından sağlam, özellikle sıcaklık iki seçenek arasında büyük farklar olabilir. Her tahlil sadece iki grup tabulating gerektirdiğinden, buna ek olarak, verileri bir basit tercih dizin olarak ifade edilebilir. Rahatlık ve kolaylık-in iki yönlü seçim deneyleri de genetik ekranlara mükellef bulunmaktadır. Ancak, büyük bir sınırlama birçok deney vahşi-türü veya mutant hayvanlar tercih edilen sıcaklığını kurmak için gerekli olmasıdır.
Degrade bir tahlil tercih edilen sıcaklık tek tahlil8' kurmak için bir fırsat sunuyor. Ayrıca, iki yönlü seçim test farklı olarak, bu hayvan, bir grup dağılımı değerlendirilmesi sıcaklıklar sürekli bir dizi ile karşı karşıya izin verir. Bir degrade tahlil bir petri ve tek hayvan kullanır ve bireysel hayvanlar10ayrıntılı davranışını karakterize için uygundur. Ancak, Petri yemekler yuvarlak olduğundan, sıcaklık bölgeleri boyutları değişir ve Merkezi'nden mesafeye göre aşamalı olarak daha küçüktür. Bu nedenle, bu kurulum nüfus hayvanların sıcaklığı seçimleri izlemek için ideal değildir.
Larva grupları sıcaklık tercihlerini değerlendirmek için uygundur sürekli bir termal degrade aparatı ve dikdörtgen bir arena istihdam burada açıklanmıştır. Aparatı oluşturmak ve montajı kolaydır. Buna ek olarak, gradyandaki doğrusal ve 42 ° C. 10 ° C arasında büyük sıcaklık aralıkları üzerinden thermotaxis değerlendirmek için kullanılabilir gibi esnektir Tahlil hızlı ve doğru sözlü gerçekleştirmek ve tekrarlanabilir veri verir. Larva tercih sıcaklığını raporlama yanı sıra, hayvan nüfusu tercihleri tek bir deneyde bir tüm doğrusal aralığında ortaya çıkarır. Bu avantajlar nedeniyle thermotaxis için gerekli genler tanımlamak için mükemmel bir seçimdir.

<table><tbody><tr><td>&lt;strong&gt;Gradient assay apparatus&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PolyScience 9106, Refrigerated/Heated 6L Circulating Bath</td><td>Thomas Scientific</td><td>9106</td><td>This model is discontinued. Updated replacement models include: 1186R00 and 1197U04 for 120 V, 60 Hz, or 1184L08 and 1197U04 for 240 V, 50 Hz.</td></tr><tr><td>Aluminum assay plate (for single directional gradient)</td><td></td><td></td><td>Outer size: 14 x 10.1 x 0.9 cm, inner size: 12.9 x 8.7 x 0.8 cm, black anodized.</td></tr><tr><td>Aluminum plate (for bidirectional gradient)</td><td></td><td></td><td>25 x 22 x 0.2 cm, black anodized.</td></tr><tr><td>Aluminum block</td><td></td><td></td><td>Outer size: 25.5 x 5 x 1.4 cm, parameters of inner channels are shown in Figure 1D.</td></tr><tr><td>Connector for aluminum blocks and tubing</td><td>McMaster-Carr</td><td>91355K82</td><td></td></tr><tr><td>Tygon Sanitary Silicone Tubing</td><td>Tygon</td><td>57296</td><td>1/4&quot; ID x 3/8&quot; OD x 1/16&quot; wall</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Items and reagents for assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pestle</td><td>USA Scientific</td><td>17361</td><td>Pestle for 1.5 mL microcentrifuge tubes</td></tr><tr><td>Thermometer</td><td>Fluke</td><td>51II</td><td></td></tr><tr><td>Thermocouple</td><td>Fluke</td><td>K type</td><td></td></tr><tr><td>Universal microplate lid</td><td>Corning</td><td>6980A77</td><td></td></tr><tr><td>35 mm dish</td><td>Corning</td><td>9380D40</td><td></td></tr><tr><td>Labeling tape (for bidirectional gradient)</td><td>Fisher Scientific</td><td>15-951</td><td>Fisherbrand labeling tape 2 in x 14 yds</td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 1% solution</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389-5KG</td><td>Prepare 18% solution right before starting assay</td></tr><tr><td>Paint brush</td><td>Fisher Scientific</td><td>11860</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Denville</td><td>C1062-P</td><td></td></tr><tr><td>Scoopula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>500 mL round wide-mouth bottle</td><td>Pyrex</td><td>1395-500</td><td></td></tr><tr><td>Cell strainer (300 mm pore)</td><td>PluriSelect</td><td>43-50300</td><td>Optional item for larvae washing</td></tr><tr><td>Cardboard box (vial tray)</td><td>Genesee Scientific</td><td>FS32-124</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Drosophila&lt;/em&gt;&lt;/strong&gt;&lt;strong&gt; food&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td>22,400 mL</td></tr><tr><td>Cornmeal, yellow (extra fine mesh,flocked) 20 kg</td><td>LabScientific Inc.</td><td>NC0535320</td><td>1,609 g</td></tr><tr><td>Brewers yeast 100 lbs</td><td>MP Biomedicals</td><td>ICN90331280</td><td>379 g</td></tr><tr><td>NutriSoy&amp;reg; Soy Flour (10 kg/unit)</td><td>Genesee Scientific</td><td>62-115</td><td>221 g</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Agar, Type II (5 kg)</td><td>Genesee Scientific</td><td>66-103</td><td>190 g</td></tr><tr><td>Karo light corn syrup</td><td>Karo</td><td></td><td>1,700 mL</td></tr><tr><td>Methyl 4-hydroxybenzoate (suspend in 200 proof ethanol)</td><td>Sigma Aldrich</td><td>H5501-5KG</td><td>72 g/240 mL</td></tr><tr><td>Propionic acid puriss. p.a.,&gt;99.5% (GC)</td><td>Sigma Aldrich</td><td>81910-1 L</td><td>108 mL</td></tr><tr><td>Phosphoric acid&amp;nbsp;ACS reagent, &amp;ge;85 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>438081-500 mL</td><td>8.5 mL</td></tr></tbody></table>

a temperature gradient assay to determine thermal preferences of drosophila larvae

Meyve sineği Drosophila melanogaster, dahil olmak üzere birçok hayvan onların tercih edilen termal manzara aramaya sağlayan çevre sıcaklığı, ayrımcılık dakika farklılıklar yeteneğine sahiptirler. Larva sıcaklık tercihleri üzerinde tanımlanmış bir doğrusal Aralık tanımlamak için biz bir sıcaklık gradyanı kullanarak bir tahlil geliştirilmiştir. Bir tek yönlü eğim kurmak için iki Alüminyum blok her biri bireysel blok sıcaklığını denetler bağımsız su hamama bağlanır. İki blok geçişin alt ve üst sınırlarını ayarlayın. Sıcaklık gradyanı plaka aralarındaki uzaklığı kapsaması su kontrollü iki blok ötede bir özel kaplamalı alüminyum levha koyarak kurulur. Su blok üst kısmında ayarlanır alüminyum plaka uçları minimum ve maksimum sıcaklık tanımlar ve bölgeler arasında doğrusal bir sıcaklık gradyanı iki blok oluştururlar. Degrade tahlil farklı yaş larva için uygulanabilir ve mutasyonlar TRP kanalları ve sıcaklık ayrımcılık için gerekli olan opsins, kodlama genler etkileyen olanlar gibi fenotipleri sergi mutantlar tanımlamak için kullanılan.

Bir 18 ° C - 28 ° C tek yönlü kurmak için degrade, biz iki su banyo sıcaklıklarında 16.8 ° C ile 31 ° c için ayarla Biz 13 puan sıcaklıklarda tüm 6 bölgeler, bölgeler arasında kenarlık çizgisi üst ve alt bölümleri içinde 26 pozisyonlarda ve özel jel yüzeyi (Şekil 2C, 2E) aşırı ucundaki sıcaklık ölçülerek elde. Sıcaklık dağılımı degrade boyunca yaklaşık doğrusal (Y 0.9672 * X + 16.19, R2 = ...

Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Burada, Drosophila larva sürekli termal degrade kullanarak tercih edilen ortam sıcaklığında belirlemek için bir iletişim kuralı mevcut.

Drosophila larva termal tercihlerini belirlemek için bir sıcaklık gradyanı tahlil

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

7.5K Görüntüleme.  University of California, Santa Barbara. Burada, Drosophila larva sürekli termal degrade kullanarak tercih edilen ortam sıcaklığında belirlemek için bir iletişim kuralı mevcut.

Video: A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Termal nosisepsiyon değerlendirilmesi Yerel ve Küresel Yöntemleri<em&gt; Drosophila</em&gt; Larva

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

Nörobilim

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Drosophila sıcaklık değişiklikler uzay ve zaman içinde performansını belirlemek için otomatik yöntemi

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Davranış

<meta charset="utf8"></head><body>DAM2 sistemleri kullanılarak Drosophila'da yüksek verimli ısı toleransı ölçümü

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

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

<meta charset="utf8"></head><body>Drosophila Activity Monitor Sistemini Kullanarak Isı Toleransı Testleri: Veri Analizi için Yürütülebilir Bir Uygulama Kılavuzu

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

Drosophila Activity Monitor Sistemini Kullanarak Isı Toleransı Testleri: Veri Analizi için Yürütülebilir Bir Uygulama Kılavuzu

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to ...

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.

Soğuk nosisepsiyon için Roman Tahlili<em&gt; Drosophila</em&gt; Larva

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

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Drosophila'da Sıcaklık Tercih Davranışının ve Sirkadiyen Ritminin Tasarımı ve Analizi

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Biyoloji

Measurement of Larval Activity in the Drosophila Activity Monitor

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

Measurement of Larval Activity in the Drosophila Activity Monitor

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

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

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

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Böceklerde Kritik Termal Limitlerin Yüksek İş Gücü Tahlilleri

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Sivrisineklerin ve diğer ektotermlerin sıcaklık tercihinin belirlenmesi

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

Drosophila larva termal tercihlerini belirlemek için bir sıcaklık gradyanı tahlil

Özet

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Bu videodaki bölümler

Termal nosisepsiyon değerlendirilmesi Yerel ve Küresel Yöntemleri

Drosophila sıcaklık değişiklikler uzay ve zaman içinde performansını belirlemek için otomatik yöntemi

Drosophila Activity Monitor Sistemini Kullanarak Isı Toleransı Testleri: Veri Analizi için Yürütülebilir Bir Uygulama Kılavuzu

Drosophila Activity Monitor Sistemini Kullanarak Isı Toleransı Testleri: Veri Analizi için Yürütülebilir Bir Uygulama Kılavuzu

Drosophila Activity Monitor Sistemini Kullanarak Isı Toleransı Testleri: Veri Analizi için Yürütülebilir Bir Uygulama Kılavuzu

Soğuk nosisepsiyon için Roman Tahlili

Drosophila'da Sıcaklık Tercih Davranışının ve Sirkadiyen Ritminin Tasarımı ve Analizi

Larvaların Faaliyet Ölçümü

Böceklerde Kritik Termal Limitlerin Yüksek İş Gücü Tahlilleri

Sivrisineklerin ve diğer ektotermlerin sıcaklık tercihinin belirlenmesi