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>接着，轻轻地梳理通过使用大小镊子或画笔幼虫以排除更大的"游荡第三龄"或小多病幼虫从被测试。含有任何不期望的遗传标记或平衡器丢弃幼虫。 </li><li>其中有排序的幼虫会放在使实验者可以用"盲目"的前资深实验室部件标签菜肴或容器perimental条件或幼虫（如果适用）的基因型。实验者可以使用由实验室部件生成的密钥实验后应用解码标签收集的数据。 </li><li>使用scoopula，转移的新鲜食品一个硬币大小的量成（35毫米×10毫米或60毫米×15mm）中清洁，标记的培养皿填充中途用室温水。 </li><li>轻轻地将排序幼虫移动到食品（防止饥饿或干燥如果测试预计将需要超过20分钟），使用镊子或画笔。 </li></ol> 2.冷探针分析<ol><li>转动冷探针单元允许其冷却至期望的温度几分钟上。如果设置为较低的温度，冷凝可能会沿探针形成。 </li><li>擦去任何多余的水分用实验室应用到幼虫之前擦拭。当不立即使用，发生在探针绝缘帽两者绝缘，并保持尖端清洁和防止损坏。 </li><li>放置一个中间第三龄幼虫到薄片暗可移动乙烯基的（典型地，用一小块从笔记本粘合剂切断）明视场显微镜下。黑色乙烯基有助于对比度可视化和在移动幼虫不接触到与探针正确地对齐。 </li><li>调整显微镜（见材料的表 ）和光单元（参见材料的表 ）至中等亮度（50-75％最大亮度），以提供对比度和防止幼虫过快干燥。 </li><li>丢弃不首先表现出正常的蠕动运动，因为它们可能混淆结果的任何幼虫。幼虫应当是湿润从水的陪替氏培养皿否则幼虫将坚持乙烯基抑制正常运动。水都不应该幼虫水坑。 </li><li>用手朝以90°的角度的前后体轴幼虫推进所述探针，使用乙烯基移动幼虫成COR矩形的位置（参见图1A）。 </li><li>东方探针的尖端轻轻划过与探针以大约45度角到显微镜载物台的幼虫的中间背侧表面铺设。 </li><li>当探针接触，启动一个定时器实验室。施加足够的向下的压力稍微缩进幼虫角质层的表面，同时仍允许向前或向后移动。 </li><li>举行的地方探头长达10秒或直到寒冷诱发的行为反应观察 - 以先到者为准。然后取出探头。 </li><li>记录观察到的行为反应和响应延迟。没有10秒内作出反应幼虫被认为是"无反应者"。延迟也可以被记录为应答者和用于测量响应的鲁棒性/振幅的变化。 </li><li>丢弃幼虫和准备下一个。 </li><li>重复步骤2.3-2.11直到达到测试幼虫的期望数目（三组的n = 20-40幼虫ùsed的这里）。 </li></ol>

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一名美国专利申请（CL）正在等候在冷探头的设计。冷探头零件编号和结构的细节的更多信息，以及在刀具设计额外的咨询会根据要求提供。

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><tbody><tr><td>Cold Probe</td><td>Pro-Dev Engineering</td><td>Custom-built on demand</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Thermal Control Unit</td><td>TE Technology</td><td>Custom Built enclosure</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Zeiss Stemi 2000 microscope</td><td>Zeiss</td><td>NT55-605</td><td></td></tr><tr><td>Fiber-Lite MI-150 High Intensity Illuminator</td><td>Dolan-Jenner Industries.</td><td>A20500</td><td></td></tr><tr><td>Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td><td>Schott North America, Inc.</td><td>Schott A08575</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>FS-1670</td><td>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><td>Paintbrush</td><td>Dick Blick Art Materials</td><td>06762-1002</td><td>Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td></tr><tr><td>35 mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>60mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Piece of black vinyl (at least 2 inches x 2 inches)</td><td></td><td></td><td>Used to provide contrast and orient larvae to the cold probe</td></tr><tr><td>Fisherbrand Scoopula Spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td>Used to move food</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>06-666A</td><td>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 ...

novel-assay-for-cold-nociception-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

Novel Assay for Cold Nociception in Drosophila Larvae

7.7K 次观看.  UT MD Anderson Cancer Center. 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。 

视频: Novel Assay for Cold Nociception in 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.

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

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.

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 ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

免疫与感染

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.

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 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.

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

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.

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 ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

测量 果蝇 拉瓦的机械结节的改进的检测和工具

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

行为学

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

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

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

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

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 ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

黑腹果蝇神经回路光遗传学作的行为测定

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Source: Chattopadhyay, A., et al. <a href="https://www.jove.com/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

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局部和全局的方法评估热痛觉

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Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

欲求联想嗅觉学习

光偏好的检测，以研究的先天和昼夜调节感光特性

单苍蝇检测方法的觅食行为的

测量果蝇拉瓦的机械结节的改进的检测和工具

温度梯度法测定果蝇幼虫的热偏好

黑腹果蝇神经回路光遗传学作的行为测定

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

新的分析冷痛觉在

摘要

探索更多视频

局部和全局的方法评估热痛觉

可视化本体感受器

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

欲求联想嗅觉学习

光偏好的检测，以研究的先天和昼夜调节感光特性

单苍蝇检测方法的觅食行为的

测量 果蝇 拉瓦的机械结节的改进的检测和工具

温度梯度法测定果蝇幼虫的热偏好

黑腹果蝇神经回路光遗传学作的行为测定

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

测量果蝇拉瓦的机械结节的改进的检测和工具