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

沙菲·萨米由内 (EY008117、EY010852)、NIDCD (DC007864、DC016278) 和 NIAID (1DP1AI124453) 提供资金支持。

1. 梯度测定设备的制造和装配设备
<ol><li>为单向梯度法制备铝化验板。<ol>
<li>用带锯和锋利的立式铣削, 将每个铝化验板 (图 1A) 从单一铝片中修剪和研磨, 其尺寸如下: 外层尺寸为 140 x 100 x 9 毫米, 内部尺寸为 130 x 90 x 8 毫米 (图 1B)。阳极处理用黑色颜料在每个化验盘内, 使幼虫更容易形象化, 防止生锈。 注: 用于单向梯度的铝化验板的制作是由一家?...

<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>

为确保本议定书的成功, 必须采取步骤, 获得足够数量的幼虫进行实验。这些包括预先喂养的苍蝇在酵母膏含瓶 2-3 d, 以改善产卵。瓶子需要放在装有水瓶的托盘中, 并封闭在一个透明的塑料袋中, 这样可以保持食物的水分, 并促进幼虫有效喂养, 同时允许接触正常的光-暗循环。然而, 酵母糊不应该是如此柔软, 苍蝇成为陷阱。每瓶雌性的数量取决于基因型。在w1118的情况下, 通常有足够...

Thermotaxis 是由移动的动物使用的选择环境以最有利情况1,2,3。如果气候过热或寒冷, 这种行为对生存至关重要。此外, 许多动物对舒适范围内的温度差异很小, 并以理想的温度寻找周围环境。这是特别重要的 poikilothermic 生物, 如果蝇, 平衡他们的体温与环境。监测幼虫 thermotaxis 的检测在识别和澄清诸如果蝇瞬态受体电位 (激进党) 通道4、5、6等分子传感器的作用方面起到了一定的帮助,rhodopsins7,8和离子型受体受体 (IRs)9, 赋予这些动物与温度敏感性在不同的温度范围。
双向选择测试提供了一种研究幼虫6,7的热偏好的方法。该化验需要建立两个不同的温度区, 并允许动物选择一侧的另一边。双向选择测试的结果可以是健壮的, 特别是当两个选项之间的温度差异较大时。此外, 由于每种化验都只涉及对两组进行制表, 所以数据可以表示为简单的偏好指数。双向选择方法的简便和简单性也适合于基因筛。然而, 一个主要的限制是需要许多实验来建立野生型或变种动物的首选温度。
梯度分析为在单一化验8中建立首选温度提供了机会。此外, 与双向选择试验不同, 它允许对一组动物的分布进行评估, 同时面对持续的温度范围。一种梯度测定法使用培养皿和单动物, 非常适合于描述个别动物的详细行为10。然而, 由于培养皿是圆的, 温度区的大小变化, 并逐渐变小取决于距离从中心。因此, 这种设置并不适合监测动物种群的温度选择。
一种连续的热梯度装置, 它非常适合于评估幼虫群的温度偏好, 这里有一个长方形的竞技场, 这里描述。该装置结构简单, 装配方便。此外, 梯度是线性的, 是灵活的, 因为它可以用来评估 thermotaxis 在大温度范围从10°c 到42°c。该检测是快速和直接的执行和产生可重现的数据。除了报告幼虫的偏爱温度外, 它还在单个实验中揭示了动物种群在整个线性范围内的偏好。由于这些优势, 这是一个很好的选择, 以确定基因所需的 thermotaxis。

<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

包括果蝇在内的许多动物都能够辨别环境温度的细微差异, 这使他们能够找到他们喜欢的热景观。为了定义幼虫在定义的线性范围内的温度偏好, 我们开发了一种使用温度梯度的检测方法。为了建立单向梯度, 两个铝块连接到独立的水浴, 每一个控制温度的个别区块。两个块设置渐变的下限和上限。温度梯度是通过将琼脂糖涂层的铝板放在两个水控块上, 使板块跨越它们之间的距离而建立的。在水块顶部设置的铝板的两端定义了最小和最大温度, 两块之间的区域形成线性温度梯度。梯度法可应用于不同年龄的幼虫, 可用于识别表现表型的突变体, 如那些有影响基因的突变的 opsins, 这是温度歧视所必需的。

为了建立一个18°c-28 °c 的单向梯度, 我们把两个水浴的温度设置为16.8 摄氏度和31摄氏度。我们通过测量在所有6个区域的上下部分的26个位置的温度, 在区域之间的边界线, 以及琼脂糖凝胶表面的极端末端 (图 2C, 2E), 获得温度在13点。沿梯度的温度分布是近线性的 (Y = 0.9672 * X + 16.19, R2 = 0.9961) (图 2E)。
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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

在这里, 我们提出了一个协议, 以确定的首选环境温度的果蝇幼虫使用连续的热梯度。

温度梯度法测定果蝇幼虫的热偏好

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

Research

JoVE Journal

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Methods to Assay Drosophila Behavior

Methods to Assay Drosophila Behavior

对检测方法果蝇行为

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

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

局部和全局的方法评估热痛觉<em&gt;果蝇</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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

欲求联想嗅觉学习<em&gt;果蝇</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 Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</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 ...

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

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

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

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

高通量检测，以检查个体产蛋首<em&gt;果蝇</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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

觅食路径长度协议<em&gt;果蝇</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 ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

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