我们要感谢Astha Maltare产生的幼虫爬行数据。我们要感谢博士尼古拉斯·兰森小的手稿给他的意见。为ALS在约翰霍普金斯大学的罗伯特·帕卡德中心（百联）和肌萎缩性侧索硬化症协会（百联）;和支持这项工作是从国家精神卫生研究院（CDN），R01MH083689。

A.幼虫爬行法 1。幼虫收集<ol><li>成立8盎司一瓶苍蝇（10-15男性10-15女性）。 </li><li>让苍蝇奠定24小时的卵子，然后明确瓶苍蝇。 （转移到​​一个新瓶的成年人，并在必要时重复）。 </li><li>孵育瓶为3-4天，或直至三龄幼虫均可见。 </li><li>添加50 - 100毫升20％的蔗糖与幼虫的瓶子，让坐了20分钟。幼虫会浮到顶端。 </li><li>收集的幼虫用25毫升血清吸管尖端切断，成网状篮子的地方。 </li><li>洗两次去离子H 2 O幼虫在网篮幼虫为实验做好准备。 </li></ol> 2。治疗药物的幼虫<ol><li>用刷子刷到5毫升的烧杯中含有一种解决方案蔗糖5％+药物输送所需数量的幼虫。 </li><li>让至少15分钟的幼虫饲料。</li><li>网状筐倒入药物治疗的幼虫和冲洗。他们现在已经准备好使用。 </li></ol> 3。运动检测（测量总距离行驶或体壁收缩） <ol><li>用刷子个别运输幼虫到： <ol><li> 15厘米的培养皿中含有超过0.2厘米2格方格纸，2％琼脂糖（以前浇，并允许变硬）。 <ol><li>计数在1分钟内越过网格线的数目。 </li></ol></li><li>良好的玻璃夹层菜含有稀释的酵母膏溶液。 <ol><li>解剖显微镜下观察，计数在一分钟内蠕动收缩（全前后运动= 1收缩）。 </li></ol></li></ol></li><li>重复，直到幼虫所需的数字已经算。 </li></ol> B.快速迭代负geotaxis（环）协议此法德scribed由加尔加诺等 5。 <ol><li>收集到一个标准的小瓶含食品（或食物+药物）新出现的光CO 2麻醉和地方的成年男性苍蝇。 </li><li>在室温下保持苍蝇（台式〜22°）为2-3天，以便从CO 2回收（稳态药物浓度的积累，如果适当）。 </li><li> 无需麻醉准备聚苯乙烯瓶约25苍蝇转让。 </li><li>苍蝇瓶组装成环装置（图1）。 </li><li>让苍蝇的环境，不受干扰15-20分钟，以适应新环境。 </li><li>在此期间，地方的数码相机〜1米（在一个平台上，如果有必要的镜头对准中旬高度小瓶中心）在前面的仪器，集中和放大到仪器的摄像头，并设置一个计时器3.0秒。 </li><li>仔细举行的环器具用你的左手，以免区URB的苍蝇，并保持用右手的计时器。 </li><li>大幅挖掘设备上的三次替补的表面，确保水龙头击倒所有的苍蝇瓶的底部是很难足够。 </li><li>同时完成第三自来水，开始倒数计时3秒。 </li><li>在三秒钟拍摄一张照片。 </li><li> 1分钟复位定时器和启动。在此期间，重置设备上的摄像头和重点，并另设三秒钟的定时器通道。 </li><li> 1分钟后，重复步骤1.7-1.10 </li><li>共5-6次试验后，上传到电脑的图像，并使用您最喜爱的图像浏览器打开，并得分的平均身高为每个小瓶攀升。 </li><li>进行统计分析，对不同群体比较，平均身高增长。 </li></ol> C.求爱和交配实验<ol><li>在早晨的第一件事，明确生产FLI瓶要使用的ES。 </li><li>在一天的课程（每3-4小时），收集新出现的性幼稚的男性和女性： <ol><li>介质的小瓶或管分别放置男性。 </li><li>每瓶/管放置5-6女性。 </li><li>在25°C下12小时的光/暗为5天的隔离收集苍蝇。 </li><li>转移到交配轮室的一名女性。 </li><li>传送一个男性的交配轮腔。 </li><li>解剖显微镜下观察，对有下列行为： <ol><li>取向（男性对女性的定向） </li><li>攻丝（男性水龙头女性的） </li><li>永歌（男延伸和振动一翼） </li><li>舔（男舔女性生殖器） </li><li>冰壶（男性卷发下腹部） </li><li>交配尝试（冰壶活动，而试图装入女） </li></ol></li></ol></li><li>观察10分钟或直至成功交配，注意到每个行为发生的时间（潜伏期），从事直到交配求偶行为（计算的求偶指数），以及对数，成功地执行一个给定的行为的总时间（频率）。 100％的野生型对一般会在5分钟内交配。 </li><li>计算 求爱指数（CI）的时间除以花求爱除以直到交配的总时间。对于野生型对这个范围通常在0.6-0.8之间。 </li></ol> D.代表结果爬行法正常野生型幼虫会徘徊〜3厘米/分钟，和展览〜40-50体壁收缩，在一分钟内。最近，我们已经制定了相关的FUS / TLS-肌萎缩性侧索硬化症，幼虫爬行缺陷，减少寿命和成人攀岩减值6果蝇模型。我们有针对性的表达Øf野生型和突变形式的FUS / TLS的运动神经元（OK-371-GAL4驱动器），并进行了幼虫爬行检测。如下图所示，野生型幼虫爬至12厘米，而野生型的FUS的表达下降约6厘米的幼虫爬行能力。表达的FUS / TLS的ALS的致病突变R521C的动物展示在他们的爬行运动非常严重受损（图1），爬行大约只有1厘米/分钟。 负geotaxis环法年轻的野生型成蝇，应该有一个3秒的时间在4-5厘米（3秒的时间可以从调整，以适应不同品系或活动水平定义为一个给定的一个给定的平均身高平均爬升高度应变/处理）。保持在底部的苍蝇，被分配一个0值。不建议使用超过25％小瓶苍蝇，因为它然后变得难以确定每一个人的苍蝇来衡量高度的位置。 ñØ脱敏已被观察到，在间隔1分钟，除了我们已经聘请了连续6个试验。关键是不重用初始数据集后，在本实验聚苯乙烯测试小瓶收集，因为到用小瓶放在新苍蝇不会爬新鲜小瓶到相同的程度。 <img alt="图1" src="/files/ftp_upload/3795/3795fig1.jpg" /> 图1。安装环检测。数码相机被放置在含有聚苯乙烯瓶苍蝇的仪器前约1米;对焦和变焦相机到设备，并设置一个计时器，以3.0秒。 <img alt="图2" src="/files/ftp_upload/3795/3795fig2.jpg" /> 图2。代表数据果蝇幼虫爬行使用检测异位表达的UAS-Fu的野生和UAS-Fu的控制下运动神经元驱动（OK371-GAL4）R521C。 <p类“jove_content”&gt;

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我们什么都没有透露。

果蝇的行为是严格受遗传和环境因素。我们和其他人，以前使用这里描述的实验，收集数据，检查飞的行为，并在果蝇 5-19仿照人类神经退行性疾病相关的基因。 第三龄幼虫爬行分析，慎重选择是关键的一步。如果用药物治疗，它会采取10-15分钟（或更取决于药物的类型和性质），以达到最大的效果，如果有良好的溶解性。因此，我们经常喂药物飞行15分钟，然后等?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>蔗糖</td><td> Fisher Scientific则</td><td> S5-500 </td></tr><tr><td>琼脂糖</td><td> Invitrogen公司</td><td> 16500-500 </td></tr><tr><td> 6盎司果蝇瓶</td><td>杰纳西科学</td><td> 32-130 </td></tr><tr><td>油漆刷（＃1） </td><td>特德·佩拉，公司</td><td> 11859 </td></tr><tr><td> 飞食品组件 </td><td></td><td></td></tr><tr><td>棒子面</td><td> Fisher Scientific则</td><td> NC9109741 </td></tr><tr><td>洋菜</td><td>杰纳西科学</td><td> 66-104 </td></tr><tr><td>糖蜜</td><td> Fisher Scientific则</td><td> NC9349176 </td></tr><tr><td>丙酸</td><td>阿库罗斯福冈</td><td> 14930-0010 </td></tr><tr><td> tegosept </td><td>顶点</td><td> 20-258 </td></tr><tr><td>乙醇</td><td>费舍尔科学的</td><td> BP2818-4 </td></tr><tr><td>酵母</td><td>杰纳西科学</td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

果蝇 ，果蝇，已用于研究的范围广泛的人类疾病，如癌症，心血管疾病及各种神经系统疾病的分子机制。我们已经优化了简单和强大的行为分析，确定幼虫的运动，成年的爬坡能力（环法），对果蝇的求偶行为。这些行为的分析是研究遗传和环境因素对飞行为的作用，广泛适用。幼虫爬行能力，能可靠地用于确定在果蝇幼虫爬行能力的早期阶段的变化，并检查他们的运动，药物或人类疾病基因的影响（转基因果蝇）。幼虫爬行检测变得更适用，如果一个基因的表达或取消导致蛹或成人阶段的杀伤力，因为这些苍蝇不活到成年，他们可以评估。这基本也可用于ssay配合明亮的光线或应力研究果蝇幼虫额外的行为反应。求偶行为已被广泛用于研究遗传基础的性行为，也可用于检查活动和协调，以及学习和记忆。 果蝇求偶行为涉及的各种感官刺激，包括视觉，听觉和化学感应信号交换之间的男性和女性，导致一系列复杂的特点以及电机最终成功交配行为。传统的成人攀岩检测（负geotaxis），单调乏味，劳动密集，耗时2-4不同试验之间的显着变化，。快速迭代5负geotaxis（环）法有许多优势，更广泛采用的协议，提供了一个重复性好，灵敏度高，高通量的方法来量化成人运动和消极geotaxis behaviors。在环法，几个基因型或药物治疗，可以同时使用大量的动物测试，高通量的方法筛选试验更适合。

Watch this Scientific Journal Video about Methods to Assay Drosophila Behavior at JoVE.com

Methods to Assay Drosophila Behavior

果蝇是一种基因和行为听话的模型系统已用于了解一个多世纪1的基础上的许多重要的生物过程的分子和细胞， 果蝇已经得到了很好的利用获得到飞行为的遗传基础的见解。

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 ...

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

42.6K 次观看.  Louisiana State University Health Sciences Center. 果蝇是一种基因和行为听话的模型系统已用于了解一个多世纪1的基础上的许多重要的生物过程的分子和细胞， 果蝇已经得到了很好的利用获得到飞行为的遗传基础的见解。

视频: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

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

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

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

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

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 ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

机械冲突避免测定测量小鼠疼痛行为

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...