Name: novel assay for cold nociception in drosophila larvae
Uploaded: 2017-04-03T15:00:00
Description: Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

我们感谢萨拉·Wu和卡米尔·格雷厄姆开发了冷探针分析，布鲁明顿果蝇库存中心飞股票，并Galko酒店实验室成员的早期阶段批判地阅读手稿。这项工作是由美国国立卫生研究院NRSA（NIH F31NS083306）到HNT，并通过NIH R01NS069828，R21NS087360和得克萨斯州MD安德森克拉克奖学金的基础研究到MJG大学的支持。

1.幼虫的制备<ol><li>提高股票或遗传杂交在25ºC培养箱中。 <ol><li>如果培养一个十字架，使用20-25处女的女性，每个含定期玉米面飞行介质小瓶15-20男性。允许女性将其转移到食品中新的小瓶中之前产卵约48小时。 </li></ol></li><li>产卵后4-5天，收集所需的基因型的3 龄幼虫，轻轻喷出的水流到糊状食物和幼虫，并倾出的内容到一个中等规模的，干净的陪替氏培养皿（60毫米×15毫米）。 </li><li>...

<ol>
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G., 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=TrpA1+regulates+thermal+nociception+in+Drosophila.">TrpA1 regulates thermal nociception in Drosophila.</a> PLoS One. 6 (8), e24343 (2011).</li><li>Zhou, Y., Cameron, S., Chang, W. T., Rao, Y. <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+directional+change+after+mechanical+stimulation+in+Drosophila.">Control of directional change after mechanical stimulation in Drosophila.</a> Mol Brain. 5, 39 (2012).</li><li>Im, S. 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此处所描述的测定可用于定性和定量评估各种遗传背景，环境影响，和/或损伤诱导的条件的幼虫伤害感受性或伤害性致敏。由于该测定法允许冷的刺激的焦点应用，用这种工具可以评价具体地在响应冷的温度周围感觉神经元的一个子集的功能。有趣的是，这些冷诱发的行为似乎利用不同类别的感觉神经元比由热7,11激活的，但完整的热伤害性电路是尚未...

果蝇已被证明是用于新颖保守的基因和背后复杂行为的神经元回路的识别非常有用的。苍蝇提供一个复杂的遗传工具包和一个简化的神经系统，其允许精确遗传和神经元操作1，2，3，4至解剖伤害感受5，6，7的细胞和分子基础。幼虫可用于这些分析是特别有用的，因为对于温和触摸8，9，10，有害热11，12，13和有害刺激4的机械感觉行为分析， </sup&gt; 11已经建立，并且透明幼虫角质层允许表皮和下面的感觉神经元的活的或固定成像。最近，有害低温的试验也已开发7，这是我们在这里详细描述。 使用细的，锥形的尖低温探针，我们表明， 果蝇幼虫表现出了一组冷特定反应性的行为，从正常运动过程中观察到的行为，以下温柔的触摸不同的，或后苛刻机械或高温的刺激7，8，11 。冷特定行为包括鲁棒的全体收缩（CT），一个45-90º加注的后段（PR）和前的同时加注以及后段成U形（US）的。这些行为的发生率随着温度的降低，但在s每个峰增加轻轻不同寒冷的气温。最近的工作表明CT响应由不同周围感觉神经元比那些对有害热或苛刻机械刺激作出反应7介导的。 很像脊椎动物伤害感受器， 果蝇多个树枝状（MD）周围感觉神经元具有arborize在表皮1复杂树枝状结构。 MD神经元存在于每个幼虫本体段，伸出的它们的轴突到腹神经索14。 MD的感觉神经元被分成基于树枝状形态四种不同的类别（I-IV）和具有不同的感觉功能4，9，10，15，16，17。而所需要的幼虫侧向车身侧倾响应IV类神经元于高温或苛刻机械刺激4，类需要III神经元温柔的触摸响应9,10和不仅由冷启动，而且还需要用于冷诱发的行为反应7。既III类和IV类神经元利用离散瞬时受体电位（TRP）通道，以促进对有害7，11，18和非有害刺激物9，10，17，19的行为反应。此外，幼虫伤害感受致敏损伤后，在细胞20和行为水平12,21。 这里所描述的试验允许quantificatio或者是正常，或潜在的n个改变行为反应冷温度范围从有害低温（≤10℃），无害的冷却（11-17℃），于环境温度下（18-22℃）。在该测定中使用的冷的温度下能够直接激活III类感觉神经元，引发健壮的，可再现的钙的增加和冷诱发的行为反应，其可以是定性和定量分析7。此测定法可应用到几乎所有的基因型的幼虫以及幼虫暴露于不同的环境条件（改变的营养，受伤，药物试剂），以确定该影响冷痛觉，疼痛致敏性或伤害性可塑性遗传和环境因素。鉴于thermosensation跨越许多物种无处不在，这种检测提供了痛觉研究中的一个有价值的工具，可能会发现新的基因目标，或将改善神经元相互作用我们的脊椎动物伤害性的理解。 定制的冷探针（见低温探针， 材料的表 ）利用控制珀耳帖装置，其冷却通过热传导的铝轴和锥形尖端的闭环温度。热敏电阻被嵌入在铝锥形尖端内报告所述控制单元上的实时温度。散热器和风扇安装于热电模块，以调节珀尔帖效应的热负荷（QC）左右（22-0℃）所需温度范围，可以实现（见热控制单元， 材料的表 ）。冷探针尖端的有害冷的刺激是通过手工施加到背中线，以链段（S）从前面和后面的端部等距离（大致段A4，参见图1A）的幼虫。为了应对寒冷刺激，幼虫通常产生三个冷诱发的行为之一的10秒内切断：一个完整​​的身体收缩（CT），一个45-90º加注前，后段的成U型（US）或后段（PR）（在结果描述）的加注。正常蠕动运动或觅食行为的过程中进行这些行为都没有。这些行为也可从温和的触摸响应和高温或有害机械刺激的厌恶轧制响应不同。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cold Probe</td>
			<td style="width:71px;">Pro-Dev Engineering</td>
			<td style="width:119px;">Custom-built on demand</td>
			<td style="width:167px;">Part numbers and construction details can be provided on request</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Thermal Control Unit</td>
			<td style="width:71px;">TE Technology</td>
			<td style="width:119px;">Custom Built enclosure</td>
			<td style="width:167px;">Part numbers and construction details can be provided on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss Stemi 2000 microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">NT55-605</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fiber-Lite MI-150 High Intensity Illuminator</td>
			<td style="width:71px;">Dolan-Jenner Industries.</td>
			<td style="width:119px;">A20500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td>
			<td style="width:71px;">Schott North America, Inc.</td>
			<td style="width:119px;">Schott A08575</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">FS-1670</td>
			<td style="width:167px;">Used to sort and handle larvae. Be sure to smooth and blunt forceps tips slightly to lower the risk of accidently puncturing or injuring the larvae</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paintbrush</td>
			<td style="width:71px;">Dick Blick Art Materials</td>
			<td style="width:119px;">06762-1002</td>
			<td style="width:167px;">Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">35 X 10 mm Polystyrene Petri Dish</td>
			<td rowspan="2" style="width:71px;">Falcon</td>
			<td rowspan="2" style="width:119px;">351008</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">60 X 10 mm Polystyrene Petri Dish</td>
			<td rowspan="2" style="width:71px;">Falcon</td>
			<td rowspan="2" style="width:119px;">351007</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Piece of black vinyl (at least 2 x 2 inches)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to provide contrast and orient larvae to the cold probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand Scoopula Spatula</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-357Q</td>
			<td style="width:167px;">Used to move food</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Kimtech Science Kimwipes</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">06-666A</td>
			<td style="width:167px;">Used to dry the larvae and cold probe if there is excess moisture</td>
		</tr>
	</tbody>
</table>

novel assay for cold nociception in drosophila larvae

生物如何感知和有害反应的温度仍然知之甚少。此外，底层的感官机械的敏化，如在患者经历外周神经病变或损伤诱导的敏化的机制，没有得到很好的表征。经遗传易处理的果蝇模型已被用于研究对伤害性热检测所需的细胞和基因， 这已经产生了多个感兴趣的保守基因。然而鲜为人知的是，在细胞和受体的有害低温感应重要。虽然， 果蝇不下去长时间暴露在低温下（≤10℃），并且将避免凉，在的行为偏好分析，他们是如何感知宁愿气温回暖，并可能避免伤害性冷刺激仅在最近被查处。 在这里，我们描述和表征第一有害低温（≤10℃）的行为分析中果蝇。使用这个工具和分析，我们表现出研究者如何定性和定量评估冷痛觉的行为。这可以正常/健康的文化条件下的疾病，受伤或过敏的情况下完成，或者可能。另外，该测定可以应用于选择用于期望的基因型，这可能会影响thermosensation，疼痛或疼痛性致敏幼虫。由于疼痛是一种高度保守的过程中，使用这个试验进一步研究热痛觉可能会搜集其他物种，包括脊椎动物疼痛过程的重要认识。

与包括偶尔的暂停，头圈，并在方向22变化的蠕动运动果蝇幼虫的举动。响应于有害低温刺激的焦点应用然而，幼虫表现出了一组独特的行为的，不像厌恶横向辊对有害热和机械刺激。这些行为是从也响应不同，以温柔的触摸8，9，10或至室温探针的触摸。冷诱发的行为如下：碾...

Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

Novel Assay for Cold Nociception in Drosophila Larvae

在这里我们展示了一个新的分析来研究果蝇幼虫冷痛觉。该试验利用能够施加焦点有害低温刺激的定制珀尔帖探头和导致量化冷的特定行为。该技术将允许冷痛觉的进一步细胞和分子解剖。

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 ...

Research

JoVE Journal

Neuroscience

Novel Assay for Cold Nociception in Drosophila Larvae

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 ...

科研

神经科学

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

A Single-fly Assay for Foraging Behavior in Drosophila

单苍蝇检测方法的觅食行为的<em&gt;果蝇</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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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 ...

Taste Preference Assay for Adult Drosophila

Taste Preference Assay for Adult Drosophila

口味偏好测定成人<em&gt;果蝇</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...