C. m. は NIDCD (DC007864、DC016278) (EY008117、EY010852)、ネイから資金援助によってサポートされて、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>

著者が明らかに何もありません。

このプロトコルの成功を確保するため、幼虫の実験を行うための十分な番号を取得する手順を取ることが重要です。あらかじめ産卵を改善するために 2-3 d 酵母貼り付け含むバイアル中のハエを餌が含まれます。バイアルは水の瓶を含むトレイに置かれ、食品の水分を保持し、通常の光サイクルへの露出を確保しながらの幼虫によって効果的な摂食を促進する透明なプラスチック袋に封入する...

モバイル動物によって温度走性を採用して、最も有利な条件1,2,3環境を選択します。過度に熱いまたは冷たい気候になった場合、この動作は生存に不可欠です。さらに、多くの動物は快適な範囲の温度の非常に小さい相違に敏感であるし、理想的な温度環境を模索します。これは、環境と自分の体の温度を平衡ショウジョウバエなど poikilothermic 生物にとって特に重要なのです。幼虫の温度走性を監視するための試金は識別し、ショウジョウバエ一過性受容体電位 (TRP) チャネル4,5,6など分子センサーの役割を明確に尽力しています。ロドプシン7、8、およびイオン型受容体受容体 (IRs)9、異なった温度較差上の温度感度を持つこれらの動物のため。
双方向の選択のテストは、幼虫6,7熱の好みを勉強する 1 つの方法を提供します。アッセイは、2 つの異なる温度ゾーンを確立することを伴なうし、動物他の上の 1 つの側面を選択することができます。双方向選択テストの結果は、堅牢な 2 つのオプションの間の温度差が大きい場合に特にすることができます。さらに、各分析には集計のみ 2 つのグループが含まれているので、データはシンプルな設定インデックスとして表現できます。使いやすさと双方向選択の試金のシンプルさは、遺伝子スクリーニングに適しています。しかし、主要な制限は、多くの実験動物の野生型や突然変異体の最寄りの温度を確立する必要は。
グラデーションのアッセイでは、単一のアッセイ8最寄りの温度を確立する機会を提供しています。さらに、双方向の選択テストとは異なり温度の連続的な範囲に直面したとき、動物の集団の分布の評価が可能です。1 つのグラデーション アッセイ ペトリ皿と単一動物を使用し、個々 の動物10の詳細な動作評価に適しています。ただし、ペトリ皿はラウンドなので温度ゾーンのサイズによって異なります、中心からの距離に応じて徐々 に小さい。したがって、このセットアップは、動物の集団の温度の選択のモニタリングに最適ではありません。
幼虫のグループの温度の設定を評価するために適しています連続熱勾配装置は、長方形のアリーナを採用し、ここに記載されています。装置は簡単に構築し、組み立てます。また、グラデーションは線形で、42 ° C まで 10 ° C から広い温度範囲で温度走性を評価するために使用することができますは、柔軟です、試金は迅速かつ簡単に実行を再現性のあるデータが得られます。幼虫の支持された温度のレポート作成に加えて、それは単一の実験で線形範囲全体に対する動物の人口の好みを明らかにします。これらの利点のための温度走性に関与する遺伝子を特定するための優れた選択肢です。

<table>
	<tbody>
		<tr>
			<td>Gradient assay apparatus</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&#34; ID x 3/8&#34; OD x 1/16&#34; wall</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Items and reagents for assay</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drosophila food</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&#174; Soy Flour (10 kg/unit)</td>
			<td>Genesee Scientific</td>
			<td>62-115</td>
			<td>221 g</td>
		</tr>
		<tr>
			<td>Drosophila 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.,&#62;99.5% (GC)</td>
			<td>Sigma Aldrich</td>
			<td>81910-1 L</td>
			<td>108 mL</td>
		</tr>
		<tr>
			<td>Phosphoric acid&#160;ACS reagent, &#8805;85 wt. % in H2O</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

ミバエショウジョウバエを含む多くの動物は、肥えた最寄り熱風景を模索することができます環境温度のわずかな違いが可能です。幼虫の温度の設定を定義すると、定義された線形範囲にわたって、温度勾配を使用してアッセイを開発しました。単一方向のグラデーションを確立するには、2 つのアルミ ブロックは個々 のブロックの温度を制御する独立した水のお風呂に接続されます。2 つのブロックは、グラデーションの上下限値を設定します。温度勾配は、プレート間の距離にまたがる 2 水制御ブロックをアガロース コーティング アルミ プレートを配置することによって確立されます。水ブロックの上に設定されているアルミ板の両端の最小値と最大温度を定義および中間の地域 2 つのブロックを形成する線形温度勾配。グラデーションのアッセイの幼虫に適用できるし、TRP チャネルと温度差別に必要なオプシン遺伝子に影響を与える突然変異などの表現型を示す変異体を識別するために使用することができます。

18 ° C から 28 ° C の単一方向を確立するには、グラデーション、2 つの水のお風呂の温度を 16.8 ° C と設定 31 ° C我々 はすべての 6 ゾーン、ゾーン間の境界線の上部と下部の部分内の 26 の位置に agarose のゲルの表面 (図 2, 2 e) の極端な両端の温度を測定することによって 13 点の温度を取得します。グラデーションの温度分布はほぼ線?...

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

ここでは、連続的な温度勾配を利用したショウジョウバエ幼虫の最寄りの環境温度を決定するプロトコルを提案する.

ショウジョウバエ幼虫の温度設定を決定する温度勾配試験

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

a-temperature-gradient-assay-to-determine-thermal-preferences

Research

JoVE Journal

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

7.5K ビュー.  University of California, Santa Barbara. ここでは、連続的な温度勾配を利用したショウジョウバエ幼虫の最寄りの環境温度を決定するプロトコルを提案する.

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

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

用いて、化合物の生理活性をスクリーニングするためのペアNanoinjectionと電気生理学的アッセイキイロショウジョウバエジャイアントファイバシステム

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

神経科学

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.

アッセイのメソッドショウジョウバエ行動

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

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.

に温痛覚を評価するためのローカルおよびグローバルメソッド<em&gt;ショウジョウバエ</em&gt;幼虫

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

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.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

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.

で食欲連想嗅覚学習<em&gt;ショウジョウバエ</em&gt;幼虫

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.

に自然免疫とサーカディアン規制Photobehaviorを勉強する光プアッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

での採餌行動のためのシングル·フライアッセイ<em&gt;ショウジョウバエ</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

視床下部細胞モデルでアポトーシスを決定するために、カスパーゼの多重化アッセイの使用

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

個々の産卵環境を調べるために、ハイスループットアッセイ<em&gt;キイロショウジョウバエ</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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.

パスの長さのプロトコルを採餌<em&gt;キイロショウジョウバエ</em&gt;幼虫

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

ショウジョウバエ幼虫の温度設定を決定する温度勾配試験

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用いて、化合物の生理活性をスクリーニングするためのペアNanoinjectionと電気生理学的アッセイキイロショウジョウバエジャイアントファイバシステム

アッセイのメソッドショウジョウバエ行動

に温痛覚を評価するためのローカルおよびグローバルメソッド

でProprioceptorsの可視化

で食欲連想嗅覚学習

に自然免疫とサーカディアン規制Photobehaviorを勉強する光プアッセイ

での採餌行動のためのシングル·フライアッセイ

視床下部細胞モデルでアポトーシスを決定するために、カスパーゼの多重化アッセイの使用

個々の産卵環境を調べるために、ハイスループットアッセイ

パスの長さのプロトコルを採餌