Name: a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster
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Description: Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

作者谨感谢布卢明顿股票中心，利群罗，查尔斯·朱克和莉莉和裕侬扬慷慨分享飞股票及以下资金:肉体-URM（以JD和SW），本科福特学院暑期奖学金（申银万国），BD公司暑期研究奖学金（以CL和DL），该蕾妮和Anthony M.马龙，MD &#39;63暑期研究奖学金（以DL），詹姆斯·&#39;75和简Colihan暑期研究奖学金（TO）通过校友/圣十字学院的家长暑期研究基金和Stransky基金会暑期研究奖学金（为TO）。特别感谢生物系和教务处在学院的圣十字对实验室全部工作的支持。

1.十字到生成MARCM苍蝇<ol><li>保持苍蝇玉米面糖蜜培养基（6.5克/升琼脂; 23.5克/升酵母; 60 g / L的玉米面; 60毫升/升糖蜜; 4毫升/升酸混合物; 0.13％Tegosept）或标准果蝇介质在22摄氏度在12小时的光照/黑暗循环。 注:比标准温度下是用来保持MARCM-股健康。这延伸到14天蛋和成年之间的发育时间。 </li><li>从一个MARCM就绪库存 - （8日龄约1）含有必要的遗传元件（GFP的上游活化序列（UAS-GFP）的GAL80阻遏蛋白，该Gal4的转录因子，的控制下使用处女雌性热休克驱动的重组，和FRT重组位点）。 注:我们MARCM现成的个股包括以下基因型:elavGal4，UAS-CD8-GFP，HS-flipase; FRT40A，tubGal80 / CYO; tubGal4 / TM3Sb。 注意:特定FRT位点，将取决于突变的位置在基因组中。例如，FRT40A用于左臂2号染色体，如NOMPC（见10的更多详细信息MARCM协议）测试突变。 </li><li>交叉MARCM就绪处女雌性到男性包含感兴趣的突变和其相应的FRT位点（例如，W-; NOMPC3，FRT40A / CYO + / +）。为了控制有效MARCM感应，设置使用雄性苍蝇含有相同FRT位点的第二交叉，但没有突变。使用5位女性的大致比例:1男在十字架。 </li></ol> 2.定时产卵<ol><li>使苍蝇交配用于至少一个晚上后，移动大人到新的小瓶中，用新鲜培养基。允许苍蝇之前再次转移杂交到新的小瓶产卵在黑暗环境中进行大约为4小时。注意产蛋期的开始和结束时间。 注意:当似乎有高数量的媒体卵经4小时interva结束之前更短的时间间隔是可以接受的湖<ol><li>保持小瓶鸡蛋足够数量（大约大于20），并存储在一个12小时的光/暗周期在22摄氏度至晚热冲击。 注意:小瓶用鸡蛋的介质上一个数低（小于大约20）是不可能产生足够数量的成蝇对于行为测试和效率起见，建议他们不被保留用于进一步的测试。 注:我们的苍蝇就提出用灯光从早上8点到晚上8点。我们已经找到了傍晚的时候分的领先优势，以更高的蛋密度，因此通常执行4之间定时十字 - 晚上8点到晚上8点 - 午夜。苍蝇奠定在室温下的鸡蛋放在一个黑暗的橱柜，然后返回到孵化第二天，以免干扰常规明暗周期在孵化器。 </li></ol></li></ol> 3.执行热休克来诱导有丝分裂重组<ol><li>大约85 - 产蛋后100小时，地点vIALS进入水浴在37摄氏度下1小时。确保水位到达媒体在小瓶的高度以上，但不完全淹没小瓶以防止幼虫溺水。 </li><li>从水浴中取出药瓶，并允许在室温下1小时的恢复期。 </li><li>地方小瓶放回37摄氏度的水浴中1小时。 </li><li>取下水浴和存储小瓶在孵化器12小时光照/黑暗周期在22摄氏度左右，直至羽化。 </li></ol> 4.准备行为测试<ol><li>羽化之后，麻醉苍蝇在冰上。选择苍蝇与包含所有为MARCM遗传元件和表示GFP表达标记的克隆很可能会根据在父个股中使用的可观察到的表型的标记物观察到的基因型。斩首苍蝇与虹膜切除术剪刀。 注:例如，我们选择了苍蝇不遗传标记CYO（大翅膀）和Sb（茬毛）来鉴定FY苍蝇，将包含所有必要创造马赛克区域的遗传元件标有绿色荧光蛋白。 注:斩首是既要防止成年人，当刺激飞走。 </li><li>放置无头苍蝇在封闭，潮湿的环境，并允许约10 - 20小时的恢复。只有用苍蝇这种权利本身扰动时进行进一步的测试。 30小时断头内的测试飞行。 </li></ol> 5.行为测试<ol><li>通过荧光解剖显微镜下观察断头苍蝇，发现标有绿色荧光蛋白的猪鬃外部感觉器官纯合子克隆。记录刷毛名称和左，右两侧。以消除任何潜在的偏见，该实验室构件进行刷毛刺激应盲目刷毛，野生型或突变体的基因型。 </li><li>通过偏转GFP引发疏导反射标塞得满满的向飞机身采用了硬毛或细镊子，观察腿再sponse。给苍蝇解除他们的腿在应对猪鬃刺激和苍蝇不动自己的腿得分为零分之一。 注:以前的研究6,7有特点的是通过刺激特定的刷毛引起腿部的反应。这包括，响应各刷毛的腿和苍蝇该特定刷毛刺激响应的比例。我们只打进了我们预期的基础上Vandervorst的分类6响应腿的动作。 </li><li>开展间隔2分钟间隔五位审判。 </li></ol>

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作者有没有竞争经济利益。

这个协议利用一个成人行为分析以筛选影响机械感觉在果蝇的突变。因为突变体的集合包含致死对元件的突变，将排除筛选作为成年人，该协议使得使用第一Lee和罗述的复杂的遗传技术的，（1999年）和详细的协议吴，罗，（2006年），以规避成人杀伤力。 MARCM诱导同源染色体之间有丝分裂重组以产生纯合细胞的克隆的区域。因为阻遏蛋白，浴盆-GAL80中有丝分裂重组发生的损失的这些?...

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table><tbody><tr><td>Brewers Yeast (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90331225&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Corn (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90141125&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Agar (1 lb)</td><td>MoorAgar Inc.</td><td>41004</td><td>Fly Food</td></tr><tr><td>Tegosept (5 kg)</td><td>Genesee</td><td>20-259</td><td>Fly Food</td></tr><tr><td>Molasses (1 Gallon)</td><td>Sugarmill Brand - Thomsen Food Service</td><td>0 2625</td><td>Fly Food</td></tr><tr><td>Propionic Acid</td><td>Fisher</td><td>A258-500</td><td>Fly Food</td></tr><tr><td>Phosphoric acid</td><td>Fisher</td><td>A260-500</td><td>Fly Food</td></tr><tr><td>Drosophila Vials, Narrow (PS)</td><td>Genesee</td><td>32-109</td><td>Fly Cultures</td></tr><tr><td>6oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td>Fly Cultures</td></tr><tr><td>Flugs - Plastic Fly Bottles</td><td>Genesee</td><td>49-100</td><td>Fly Cultures</td></tr><tr><td>Rayon Balls, Large</td><td>Genesee</td><td>51-100</td><td>Fly Cultures</td></tr><tr><td>Droso-Filler, Narrow</td><td>Genesee</td><td>59-168</td><td>Fly Food Preparation</td></tr><tr><td>Droso-Filler, Bottles</td><td>Genesee</td><td>59-170</td><td>Fly Food Preparation</td></tr><tr><td>8A-C / gear driven lab stirrer with c-clamp mount&amp;nbsp; 1/15 HP, 700 rpm variable speed, 115 V, 50/60 Hz</td><td>Cleveland Mixer</td><td>8A-C</td><td>Fly Food Preparation</td></tr><tr><td>Water jacketed Kettle</td><td></td><td></td><td>Fly Food Preparation</td></tr><tr><td>Diurnal Growth Chamber</td><td>Forma Scientific</td><td></td><td>Temperature and light/dark cycle controlled</td></tr><tr><td>Water bath</td><td>VWR</td><td></td><td>For heat shock</td></tr><tr><td>MicroScissors&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;15000-08</td><td>For removing heads</td></tr><tr><td>Fluroscence Dissecting Microscope</td><td>Zeiss</td><td>SteREO Discovery V8&amp;nbsp;</td><td>With GFP cube (KSC295-814D) band pass filter</td></tr><tr><td>Fluroscence Light Source</td><td>Zeiss</td><td>X-Cite 120</td><td>Fiber optic light pipe makes this easy to configure&amp;nbsp;</td></tr><tr><td>Camera for Scope</td><td>Zeiss</td><td>AxioCam ICc1</td><td></td></tr><tr><td>Image acquistion software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td>for cold anthesia</td></tr><tr><td>Homemade cold anthesia tray</td><td></td><td></td><td>for cold anthesia decapitation</td></tr><tr><td>Plastic boxes</td><td></td><td></td><td>for recovery of decaptitated flies in humid environment</td></tr></tbody></table>

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

因为结构和功能同源性的哺乳动物内耳的毛细胞，即支配果蝇外部感觉器官的神经元提供机械感觉的研究的优良模型系统。这个协议描述在果蝇中，可用于识别与机械感觉干扰突变简单的触摸行为。一个macrochaete的触觉刺激刷毛上的苍蝇的胸部引出一清洁反射从第一或第三站。与机械传导（如NOMPC）干扰的突变，或者与反射弧的其他方面，可以抑制疏导响应。成人行为的传统画面就会错过这一发展过程中发挥重要作用的突变体。相反，该​​协议结合镶嵌分析与阻遏细胞标记物（MARCM）触摸屏，以允许纯合突变细胞中，只有有限的区域由EXPR生成并标分裂国家绿色荧光蛋白（GFP）的。通过测试MARCM克隆为异常的行为反应，有可能筛选的致死对元件的突变的集合以寻找涉及机械感觉新基因，将已经错过由更传统的方法。

此协议的成功在很大程度上依赖于可检验的苍蝇的总产率，热休克的有效性以诱导有丝分裂重组，并获得在果蝇以2分钟的间隔刺激一个健壮的行为反应的能力。鉴于胚胎的估计12.5％包含必要MARCM元素和成年人E关闭对GFP表达的潜在的估计的16.67％，这是关键的期间定时规定以获得高数鸡蛋。一个成功的定时的一个例子笼罩，将在该协议的其余部分被用来显示在?...

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

a-behavioral-assay-for-mechanosensation-marcm-based-clones-drosophila

Research

JoVE Journal

Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

10.2K 次观看.  College of the Holy Cross. In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

视频: A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

神经科学

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.

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 ...

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 ...

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

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

测试<em&gt;果蝇</em&gt;用Y型迷宫测定嗅觉

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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.

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 ...

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 ...

一个行为分析对机械感觉MARCM为基础的克隆

摘要

探索更多视频

此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

对检测方法果蝇行为

局部和全局的方法评估热痛觉

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

测试

通过计算机跟踪检测大鼠嗅觉行为

新的分析冷痛觉在

黑腹果蝇神经回路光遗传学作的行为测定

一个行为分析对机械感觉MARCM为基础的克隆

摘要

探索更多视频

此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

对检测方法果蝇行为

局部和全局的方法评估热痛觉

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

测试

通过计算机跟踪检测大鼠嗅觉行为

新的分析冷痛觉在

黑腹果蝇神经回路光遗传学作的行为测定

测量果蝇拉瓦的机械结节的改进的检测和工具