柑橘研究与发展基金会提供资金确认。感谢天使霍伊特和迈克尔·弗洛雷斯进行生物测定。

<ol><li>组件的定制设计，两端口除以T-嗅觉Mann 等人先前所描述的10，在图1中示出。嗅觉仪包括一个30厘米的玻璃管中，分叉成两个单独的端口有聚四氟乙烯带，形成一个T-迷宫。每一半是类似于2-的选择样式嗅觉使受验者之间选择两个潜在的气味字段的臂。 <ol><li>大会和随后的检测应该发生在一个温度控制的房间或环境室。 </li><li>垂直贴装玻璃T-迷宫嗅觉仪如在图1中所示，并将其定位在萤光灯下的900勒克斯灯泡。 </li><li>为了获得最佳的光扩散，大会应安装在一个1.0×0.6所述0.6米不透明的容器中，以最大限度地提高光扩散均匀。 </li><li>嗅觉武器将被安置在固相微萃取 - 持有/气味源可人情事可赞可叹的气味源，ER（SPMEC）（ARS公司，盖恩斯维尔，佛罗里达州）特氟纶，玻璃管接头。每个SPMEC是由支持对传入和传出的空气流（ 图1）的入口和出口阀的直玻璃管（17.5厘米长×3.5厘米宽）。 </li><li>的空气输送系统连接到外部信号源的加湿和碳 - 纯化的空气，是在恒定压力下（如果外部源不可用的空气输送系统具有用于空气净化的内部碳过滤器和用于加湿的空气鼓泡）。 </li><li>所需的化学样品溶解在适当的稀释剂（溶剂）和吸液管到一个缓慢释放的基材，如棉灯芯（小资​​约翰包装公司，位于北卡罗来纳州Concord）。控制治疗应包括仅与溶剂浸渍棉灯芯。 </li><li>为了防止重大污染的玻璃器皿，包络治疗组和对照的棉灯芯在实验室组织（Kimwipes，金佰利，罗斯威尔，GA）和分为两个SPMEC的武器（ARS公司，盖恩斯维尔，佛罗里达州）的地方。以活植物样品的情况下，一个断头台挥发性收集室可以连接到包含地方的SPMEC（ 图1）的治疗的嗅觉。 </li><li>提供纯化和通过SPMEC（或断头台挥发性室）通过连接到一个空气输送系统（ 图1）的两个泵的加湿空气。气流速度保持在0.1升/分钟通过这两种武器的嗅觉。 </li></ol></li><li>用人的嗅觉来测试节肢动物公认的驱虫剂。在本说明书中，半翅目昆虫，亚洲柑橘木虱Diaphorina螨 ，被用来作为测试节肢动物。响应进行测试，以单独或组合使用具有驱避剂（二甲基二硫化物）和清洁空气作为气味来源的柑橘挥发物。 <ol><li>进行所有的实验，在标准温度和相对湿度。 </li><li>在此之前的所有测试，暴露吨EST受试者清洁空气与清洁空气或溶剂与清洁的空气中的T-迷宫嗅觉来验证的情况下的位置的偏置或溶剂对测试对象的行为的效果的。 </li><li>随机分配的气味源的武器的嗅觉开始的每一个生物测定。扭转这个位置后，至少每10个考试科目检测，以消除潜在的位置偏差。 </li><li>测试30（120），每次治疗相结合的考试科目的最低响应。 </li><li>允许考试科目100-300秒以前建立的响应（潜伏期）的时间间隔的基础上表现出的行为反应。 </li><li>记录如果拍摄对象进入治疗组，对照组的或保留的释放口或T-迷宫部门的在下面。 </li><li>比分治疗或控制臂的选择，当测试对象在T-迷宫分为嗅觉和移动手臂穿过部门。 </li><li>得分释放臂的选择时，一个测试ubject仍然在发布的端口或以下的T-迷宫部门。 </li><li>的清洁的嗅觉和连接管，用2％的肥皂溶液彻底和烘烤的玻璃成分，在200°F之间的使用不同的气味处理。 </li><li>对于在其中假定斥治疗在清洁的空气的化学治疗与带或不带T-迷宫嗅觉测定，比较在释放点剩余的测试对象的数目，而不是输入处理之间的嗅觉仪由一个单向方差分析（ ANOVA）其次通过Tukey的HSD检验（α&lt;0.05）。在案件中，考试科目离开的释放臂，比较，选择控制臂与治疗组与卡方（χ2）分析在α&lt;0.05。 </li></ol></li></ol>

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笔者收到的柑橘研究与发展基金会提供的资金。

这里，用于测量小节肢动物（昆虫纲：半翅目：木虱科），加臭剂的反应的测定装置和协议描述。该方法涉及一个选择的测试，使昆虫之间作出选择的分析评估假定引诱的情况下气味领域。此外，受验者可以显示三个类型的行为离开的释放臂和输入的任一个的两个潜在的气味字段，或剩余的释放臂中，在的情况下，拒检测。嗅觉允许高吞吐量的数据收集，因为它利用的负面geotactic的（趋势向上移动）和正趋光性（趋势走向光明）许多节肢动物的自然行为反应。虽然本演示使用一个木虱测试的例子，该法可以很容易地适应节肢动物广泛，无论是使用飞行或步行的交通为主导的模式。 12。具体这种olfactometers设计千差万别，然而，这里所描述的双选择检测的一般主题的变化，以及，类似的Y型管检测已被经常用来测量节肢动物响应于化学品。突破性的数据收集阐明昆虫飞行和方向的信息化学物质的基本机制，以及数据的通知害虫控制工具的实际使用也导致较大的飞行通道，可能会导致持续飞行的昆虫9。 通常是必要的定制设计的嗅觉和相关仪器的特定节肢动物测验对象的生物学。这些olfactometers，可用于一般的昆虫之间是有用的，比规格IFIC向一小群，有时一小群虫的经济意义决定了一个非常具体的嗅觉和检测技术发展的需要。目前描述的设计建立在以前熟悉的节肢动物技术。它允许一个更标准的双选择测定比Y型管，其中的生物测定实验舞台或选择测试需要一个Y形的玻璃装置7的形式。通常情况下，这样的Y型管的一个臂，将接收的处理的加臭剂，而其他的将被留为空白13。的变化上等olfactometers，包括增加多个辐射武器14，甚至另外的土壤介质中的生物移动通过土壤15测定的行为。在显影此类olfactometers和相关检测，它是重要的是要考虑如何接近自然条件被复制，从而在测试受试者的生物学方面的评估的行为反应的真正的相关性。到点菜RGE程度，收集到的数据将被视为有用的行为生物测定的相关性，与生物体的行为生态学16。 目前描述的是专门设计的，往往会引发短“跳”16飞行一个半翅目昆虫的嗅觉和行为生物测定。的垂直方向的嗅觉和光源安排方便昆虫运动开始，从而随后的化学介导的取向行为可以有效地进行测定，并具有高吞吐量。这种行为有可能被用于试验的安排可以广泛的飞行或步行节肢动物类群，或者也可以很容易地修改，以适应需要的非节肢动物生物。

节肢动物（包括昆虫和蜘蛛）通常被定义为害虫，因为它们要么与人类食用的竞争在农业或城市设置或传播疾病的病原体的因果关系。大量的投资，这些害虫的控制工具的发展。这些工具可以有很大的不同，并已经取得了显著的进展在发展无公害工具，修改或利用害虫的自然行为与使用行为的引诱或驱虫剂2,3。 节肢动物害虫往往依赖于的嗅觉信号或线索队友和/或主机位置4。因此，发现有效的害虫引诱或驱虫剂，常可导致控制工具的发展。这个发现的过程包括几个步骤。首先，测试对象的自然行为必须理解。例如，一个人必须确定是否一个给定的物种的一个性别吸引相反寻找配偶的性生活。如果是这样，人们必须确定是否介导的这种吸引力是一种化学品，化学品生产。进一步的步骤包括：确定化学品生产，并最终识别。整个过程中，受验者的（昆虫或螨虫）的行为反应，加臭剂必须进行测试。最初，将被测试的测试对象的行为的天然物质。例如，一个雄蛾的响应可以被测试到其女性同种的性信息素腺体的提取物。另外，草食动物及其寄主植物的自然气味的反应进行测试。虽然这些调查往往有一个切合实际的重点发展的管理工具，5,6，它们也可以被应用到研究，解决根本的问题，如形态7。后续的步骤包括测试节肢动物的反应鉴定天然产品合成的。 ünderstanding节肢动物是如何回应的化学品需要制定适当的工具来衡量自己的行为。通过这些工具产生的数据将取决于研究者能够测量自然的行为在受控制的环境中。超过六十年前，的持续飞行olfactometers和隧道测量昆虫的气味分子反应8,9。 Olfactometers在设计显着变化，往往是针对特定的昆虫或正在调查的问题。这篇文章的目的是描述的设计和使用的垂直方向，T-迷宫嗅觉测量昆虫气味分子（ 图1）。利用嗅觉的垂直方向的许多昆虫表现出负趋地性（趋势向上）。 A光源正上方的嗅觉优势，积极展出的许多昆虫的趋光性（趋势走向光源）。该olfactomETER是特别有用的半翅目昆虫，往往不表现出运动容易在水平取向olfactometers和，有很大的影响的光强度相对于取向。然而，这种装置可用于范围广泛的飞行和爬行昆虫，以及蛛形纲动物。嗅觉可以评估引诱剂和驱虫剂。引诱剂的情况下，分叉气味字段（ 图1）建立，使用假定的诱食释放一侧上的相对侧上与清洁的空气释放。选择记录时，昆虫在基地的嗅觉发布选择的气味的领域中的一个。在的情况下，驱避剂，三分之一的行为是可能的。除了向上运动分为两个气味字段，缺乏向上运动可以被记录表示无响应的刺激或击退无论从气味刺激字段。

<table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td>两个端口分为T-嗅觉</td><td>分析研究系统（ARS），佛罗里达州盖恩斯维尔</td><td>无（定制） </td><td>如果无法从ARS，南方科技（盖恩斯维尔，佛罗里达州）目前也建立和分发等设备。 </td></tr><tr><td>固相微萃取控股/气味源室，特氟纶，玻璃管接头的。 </td><td>分析研究系统（ARS），佛罗里达州盖恩斯维尔</td><td> RV-R3 </td><td>请参阅上述评论</td></tr><tr><td>空气输送系统</td><td>分析研究系统（ARS），佛罗里达州盖恩斯维尔</td><td> HADS-2AFM2C.4 </td><td>请参阅上述评论</td></tr><tr><td>断头台室</td><td>分析RESE弓系统（ARS），佛罗里达州盖恩斯维尔</td><td> L3GP3 </td><td>请参阅上述评论</td></tr></tbody></table>

vertical t-maze choice assay for arthropod response to odorants

由于经济的重要性，害虫的农作物，城市环境，或为载体的植物和人类的疾病的昆虫和蜘蛛，各种技术正在开发作为控制工具。这些工具的一个子集，主要修改节肢动物吸引或排斥的行为。因此，节肢动物往往是行为调查的重点。已经开发的各种工具，来衡量节肢动物的行为，包括风洞，飞行工厂，servospheres和各类olfactometers的。这些工具的目的是衡量昆虫或节肢动物的视觉或更频繁的嗅觉。的的的垂直T-迷宫oflactometer描述的这里措施昆虫引诱或驱虫剂进行选择。它是一种高通量的检测设备，充分利用积极的趋光性的吸引力，光展出的许多节肢动物和负趋地性（趋势向上走或飞）。该olfactomet呃由一个30厘米的玻璃管中，被分为两半，有聚四氟乙烯带，形成一个T-迷宫。每半个作为一个手臂的嗅觉，使做出选择的考试科目在两个潜在的气味分析领域中涉及引诱。缺乏正常响应已知的引诱在涉及的驱虫剂的试验中，也可以被测量作为第三个变量。

如图2A所示景点的亚洲柑橘木虱（Diaphorina柑桔 ）的成人产后寄主植物挥发物（柑橘类）的气味。显著（α&lt;0.05），更多的成年人选择的手臂从生活的柑橘类植物的气味的T-迷宫嗅觉，较空白（清洁空气）控制。 在图2B中示出一个例子的斥力。在这种情况下，昆虫暴露在一个臂的T-迷宫接收柑橘挥发物，而第二臂收到从柑橘类的挥发物进行处理的植物，与公知的驱避剂，二甲基二硫化物11。在这种情况下，观察到三种类型的行为。木虱被捕，释放臂，而不是响应统计学上没有显着差异从数进入臂接收从柑橘类植物（ 图2B）的气味的数目。然而，没有木虱进入了手臂，含叔他寄主植物挥发物时共同提出的驱蚊（ 图2B）。 <img alt="图1" src="/files/ftp_upload/50229/50229fig1.jpg" /> 图1。示意图的垂直取向的T-迷宫嗅觉仪连接到空气输送系统和气味处理释放装置，该图是一个适应Mann 等人的 图1中的10。一个测试对象单独被放置到释放腔的2端口除以T-嗅觉。走向的气味，它允许控制的向上运动到嗅觉和防止昆虫从释放点区域爬放回通过闸阀。管分叉10厘米，门后的值。积极应对的气味记录一旦昆虫移动0.5厘米，到一个特定的部门。的断头台挥发性收集室可用于代替的固体PHASË微萃取 - 持有/气味源室（SPMEC）的为气味源。 <img alt="图2" fo:content-width="3.5in" fo:src="/files/ftp_upload/50229/50229fig2highres.jpg" src="/files/ftp_upload/50229/50229fig2.jpg" /> 图2。响应的亚洲柑橘木虱（Diaphorina柑桔 ）的成人柑橘寄主植物与清新的空气控制（A）或柑橘类植物挥发物对柑橘挥发物的挥发物联合发布的驱避剂（二甲基二硫醚）（B）在垂直方向的Ţ迷宫嗅觉仪。 图2A：单向方差分析（ANOVA），随后通过Tukey的HSD检验（α&lt;0.05）进行比较的数目木虱任臂（2个处理组中，n = 120）之间作出选择。列由不同字母表示显着不同于彼此（α&lt;0.05） 图2B：单向分析产品是Tukey的HSD检验（α&lt;0.05），其次是方差分析（ANOVA）进行比较木虱的数量选择ARM或留在释放点（3个疗程，N = 120）的。由不同的字母表示的列是显着不同的另一个（α&lt;0.05）。

Watch this Scientific Journal Video about Vertical T-maze Choice Assay for Arthropod Response to Odorants at JoVE.com

Vertical T-maze Choice Assay for Arthropod Response to Odorants

垂直，T-迷宫的嗅觉测定的节肢动物的行为反应。嗅觉让实验者衡量选择考试科目进行时，受到两个潜在的气味领域。与此装置可以测量从气味分子的吸引力和排斥力。

的垂直T-迷宫的选择含量，节肢动物响应加味剂

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

vertical-t-maze-choice-assay-for-arthropod-response-to-odorants

Research

JoVE Journal

Neuroscience

14.6K 次观看.  University of Florida. 垂直，T-迷宫的嗅觉测定的节肢动物的行为反应。嗅觉让实验者衡量选择考试科目进行时，受到两个潜在的气味领域。与此装置可以测量从气味分子的吸引力和排斥力。

视频: Vertical T-maze Choice Assay for Arthropod Response to Odorants

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

科研

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 ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification (RFID) readers built into artificial flowers that display various visual cues, and RFID tags (i.e., passive transponders) glued to the thorax of bumblebee workers. The novelty in our implementation is that RFID readers are built directly into artificial flowers that are capable of displaying several distinct visual properties such as color, pattern type, spatial frequency (i.e., &#8220;busyness&#8221; of the pattern), and symmetry (spatial frequency and symmetry were not manipulated in this experiment). Additionally, these visual displays in conjunction with the automated systems are capable of recording unrewarded and untrained choice behavior. The second method consists of recording choice behavior at artificial flowers using motion-sensitive high-definition camcorders. Bumblebees have number tags glued to their thoraces for unique identification. The advantage in this implementation over RFID is that in addition to observing landing behavior, alternate measures of preference such as hovering and antennation may also be observed. Both automation methods increase experimental control, and internal validity by allowing larger scale studies that take into account individual differences. External validity is also improved because bees can freely enter and exit the testing environment without constraints such as the availability of a research assistant on-site. Compared to human observation in real time, the automated methods are more cost-effective and possibly less error-prone.

This video describes Radio-Frequency Identification (RFID) and motion-sensitive video recording methods to monitor choice behavior by bumblebees.

无线射频识别和动作感应视频高效地自动Unrewarded选择行为的记录由大黄蜂

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

电生理记录<em&gt;果蝇</em&gt; Trichoid Sensilla响应低挥发性气味

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

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 ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

脑干中的数据采集与分析 在小鼠中引发响应音频测量

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...