笔者想，感谢Erin Fennern的协助实验。这项工作是由挪威研究理事会（180504，185306和191699），皮尤慈善信托基金，国家衰老研究所（NIA P01 AG22500）。

第1部分:RNAi介导的双基因敲除 1。双链RNA合成<ol><li>设计针对VG，USP和控制基因编码绿色荧光蛋白（GFP），这是不是在蜜蜂基因组的双链RNA引物设计引物，利用网上免费软件引物（ <a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a> ） 。 </li><li>合成的双链RNA为VG，USP和GFP:利用RiboMax T7 RNA生产系统在体外转录自Promega。 </li><li>双链RNA纯化: <ol><li>变性和复性:热的dsRNA到85°C为5分钟，让它冷却下来，在室温下1小时。 </li><li>的DNase I处理:加1微升的DNase I到每个dsRNA的反应，并把它们通过轻弹管。在37℃下孵育15分钟</li><li>加入150μl无核酸酶的水和750微升TRIZOL - LS的每一个反应，轻轻混匀投资耳听管。孵育样品在30℃下5分钟</li><li>添加到每个样品200μL氯仿。大力混合，持续20秒。离心样品在4℃下以12,000×g的15分钟</li><li>将上清转移相到每一个新的管中，并添加到各管中加入500μl异丙醇。将它们混合颠倒离心管。孵育混合物于-20℃下的20分钟离心机在4℃下以12,000×g的10分钟</li><li>去上清，使用1,000微升75％乙醇洗涤沉淀。后5分钟，以7500×g离心管在4℃，空气干燥的双链RNA沉淀，用无核酸酶的水，以溶解沉淀。为了保证疗效的基因的下调，双链RNA的浓度应为约9-10微克/微升。 </li></ol></li></ol> 2。腹腔注射双链RNA 双基因敲除，有两种策略:1）单注:混合两种基因的双链RNA和仁济克拉的混合物，2）双天注射剂:注射针对一个基因的第一天的第一个双链RNA，并注入第二双链RNA，在第二天到相同的蜜蜂。 <ol><li>冰寒新出现蜜蜂在4℃冰箱1-2分钟。当蜜蜂被固定，安装3-4蜜蜂在培养皿昆虫针其腹部和thoraces之间越过固体蜡并行。 </li><li>再次在4℃冰箱冷藏蜜蜂。确保蜜蜂是完全不动。它需要大约1-2分钟。蜜蜂看起来应该松动。如果蜜蜂变得卷曲或扭曲，这意味着他们都是冰鲜太长，应加以避免。冷太多造成很高的死亡率，但完整的固定是非常重要的，因为任何运动伤口的大小和死亡率增加。 </li><li>汉密尔顿微量注射器，将一次性30 G针（BD）。 3微升的双链RNA，并确保在注射器中有没有气泡。针被插入到侧腹部，以避免损坏晋内脏。按注射器柱塞慢慢地使被吸收的双链RNA。由于双链RNA非常粘稠，需要2-3秒被完全驱逐出注射器。完全倒推柱塞后，留下的针在伤口上，持续4-5秒。观察蜜蜂注射后3-5秒。如果从伤口的血淋巴滴泄漏，丢弃的蜜蜂。 </li><li>使用不同的颜色来标记他们的thoraces对应不同的治疗方法。此时如果使用单次注射战略，蜜蜂可以被放置到一个殖民地1小时后观察。但是，如果使用两日的喷射策略，在被注射与第一次的dsRNA，蜜蜂应放置在气缸内的网笼的一侧供给的蜂蜜，并保持在孵化器在34℃和80％湿度。 </li><li>第二天，按照步骤2.2和2.3寒意在笼蜜蜂，它们安装在蜡板。进行注射，因为它是与第二双链RNA的说明步骤2.4的床。 </li><li>让蜜蜂在室温下恢复1小时，并介绍他们到一个殖民地。 </li></ol>注意:击倒效果可以由定量RT-PCR（实时定量RT-PCR）和免疫印迹检测。在一般情况下，击倒效果各不相同，取决​​于很多因素。在我们的研究中，可以检测单一注射后第7天，为期两天的注射双基因敲除。 第2部分:长鼻扩展响应（PER）检测 1。收集蜜蜂从蜂巢注射后第6天，收集处理后的蜜蜂从殖民地，而不吸烟者。收集蜜蜂金属网筒笼。保持每笼3只蜜蜂。 2。安装蜜蜂为每冷藏的蜜蜂在4°C，直到他们完全固定。在每只蜜蜂垂直安装成一个塑料管，专为市盈率。全光照，保持管内的腹部G两个小块胶带，保持头部伸出的管子。将机架上有两排小塑料柱管，并分配一个ID号每个管（蜂）。成一个孵化器（34°C和80％的湿度），然后把处理后的蜜蜂在机架上，并让他们在孵化器2个小时。 3。准备蔗糖水解决方案系列准备0％，0.1％，0.3％，1％，3％，10％和30％的蔗糖水溶液和加载的成15-20ĝ针的注射器的解决方案。 4。每次试验 0％的溶液（水），然后通过0.1％，0.3％，1％，3％，10％和30％的蔗糖水溶液液滴的触摸蜜蜂的天线。测试所有的蜜蜂先用清水允许使用各解决方案之间的间隔至少两分钟。因此，我们通常至少有20蜜蜂每次试验。如果蜜蜂完全伸出长鼻，积极响应计算。 5。数据管理总结每一个人的积极响应，味觉反应得分（GRS）。进行适当的统计分析数据。 第3部分。基因击倒验证的脂肪体解剖从7日龄治疗的蜜蜂另一套。程序是用一个标准的TRIZOL RNA提取，随后通过DNA酶I处理24。Vg和美国药典的基因表达分析通过两个步骤的实时定量RT-PCR。 肌动蛋白被用作参照基因，因为它具有在不同组织中的表达稳定蜜蜂。发布时间实时PCR vg的和美国药典基因的引物，用于在本实验中24。数据分析使用三角洲三角洲CT方法25。

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We have nothing to disclose.

我们的研究是第一次努力，同时打掉两个基因在成虫。我们的研究结果表明，这两种策略（单次注射，为期两天的注射双链RNA注射）双基因抑制，同时基因沉默的VG和USP是有效的，可以测试后第6天的双链RNA注射到蜜蜂8,9。然而，基因敲除的功效不同的靶基因，蛋白质周转率和双链RNA的细胞或器官的吸收效率取决于转录水平不同的昆虫种类。此外，RNA干扰的效果是剂量依赖性?...

RNA干扰（RNAi）是基于RNA的转录后基因沉默，这发生在各种各样的真核生物。 RNAi过程被触发时由内源性或外源性的双链RNA（dsRNA）的前体。双链RNA激活核糖核酸酶蛋白Dicer酶结合并切割双链RNA小片段（20-25个基点）。然后，小片段的双链RNA导Argonaute蛋白的mRNA互补的识别和切割RNA诱导的沉默复合物（RISC），催化剂组分。在哺乳动物中，超过30 nt的双链RNA，激活抗病毒反应（IFN）干扰素反应，从而导致非特异性降解RNA转录2。然而，长dsRNA已被证明是有效的，具体的昆虫，因为有一个缺乏这IFN 3。 长双链RNA下调靶基因已被用于在不同的昆虫种类。蜜蜂是一个π介子能效比昆虫生物，其中许多重要功能基因的开发和行为已被揭露出来使用双链4,5。几个dsRNA的交付方法，已进行了蜜蜂的dsRNA喂养蜜蜂幼虫6有效下调靶基因的表达，而在蜜蜂胚胎和成年蜜蜂7,8双链RNA注射液是一种有效的基因敲除的方法。 展出的昆虫双链RNA的基因敲除的影响是短暂和局部的。有研究表明，腹腔注射双链RNA和胸双链RNA注射，有效地抑制靶基因的表达，腹部脂肪体细胞的昆虫9,10。双链RNA注射到腹腔和胸腔和脂肪体细胞能够采取双链RNA细胞血淋巴沐浴10。然而，基因在其他器官，如卵巢和大脑，不能有针对性要么腹腔或胸腔注射。以目标基因在蜜蜂的大脑，大脑的dsRNA注入也被执行，有效地影响局部脑区11靶基因的表达。在这里，我们只记录腹双链RNA注射，这是较为常用的成年蜜蜂。 RNA干扰已被主要用于针对一个单一的基因，一直是一个强大的工具来揭示基因功能。然而，任何基因从别人不是孤立的，它是在复杂的调控网络。了解生物过程的一个关键是解剖与对方，这需要同时操作多个基因，而不是一个单一的基因敲除基因如何相互作用。在哺乳动物细胞系同时抑制两种或三种基因，科学家们已经成功地通过输送系统12多的microRNA（miRNA）的发夹设计13。但在昆虫，多基因击倒仍未经验证。这里，我们PRESEN了吨不同注射的策略可以实现双基因敲除。我们的目标是两种基因:卵黄蛋白原（VG），它编码一个卵黄蛋白的前体，和ultraspiracle（ 美国药典 ），保幼激素（JH），编码一个假定的受体可能作为一种转录因子介导的反应在蜜蜂JH 14。 Vg和JH相互调节在反馈回路15，并参与了蜜蜂行为调节9。使用双基因敲除，我们扰乱Vg和JH途径，并发现它们是如何共同影响蜜蜂的行为和生理VG，USP和JH如何互动9。 味觉感知是一种行为预测蜜蜂社会行为16。行为的发展，巢蜜蜂高味觉感知行为成熟的快，通常在生命早期的牧草，喜欢收集花粉16,17。虽然监管米相关echanisms味觉感知目前仍不清楚，有研究表明，味觉的感知与内部能量代谢9，荷尔蒙分泌18,19和生物遗传胺通路20。 Vg和JH重要的荷尔蒙调节调节味觉感知7,21。在实验室中，味觉感知蜜蜂的变化进行评估测试长鼻延伸反应（PER）的不同的蔗糖溶液。每个蜂测试升序一系列浓度为0.1，0.3，1，3，10，30％的蔗糖的液滴的水通过触摸她的触角。注意，如果蜜蜂完全延伸她的长鼻液滴的水或蔗糖时，每个天线被触摸到的正响应。理想的反应溶液的数量的基础上，每一个人的味觉感知水平，可确定16。但是，应用程序的PER并不限于测量克感知ustatory。市盈率也是一种有效的方法来测试，如饱食与饥饿的代谢状态的蜜蜂。蜜蜂蔗糖有较大的反应一般是饿（王Amdam的，未发表的数据）。此外，市盈率的模式也可以被用来在联想学习和记忆的蜜蜂。在这种情况下，蜜蜂将接受培训，蔗糖水与气味是否存在关联。当蜜蜂学习的联想，只有存在的气味能唤起积极长鼻响应，没有奖励他们与蔗糖22,23。在这段视频中，我们将展示如何执行市盈率评估味觉感知，已与VG和USP双击倒在先前的研究9。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>GE Healthcare PCR beads in 96 well plate</td>
			<td>GE Healthcare</td>
			<td>27-9557-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboMax T7 RNA production system</td>
			<td>Promega</td>
			<td>P1300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trizol LS</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroform:Isoamyl alcohol 24:1</td>
			<td>Sigma-Aldrich</td>
			<td>C0549</td>
			<td></td>
		 </tr>
		 <tr>
			<td>isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hamilton micro syringe</td>
			<td>Hamilton</td>
			<td>80301</td>
			<td></td>
		 </tr>
		 <tr>
			<td>30G BD disposable needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30960</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereo dissection microscope</td>
			<td>Leica Microsystems</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)

此视频演示新颖的RNA干扰（RNAi）技术下调两个基因同时使用双链RNA（dsRNA）的注射液的蜜蜂。同时还介绍了协议的长鼻扩展响应（PER）检测味觉感知测量。 RNAi介导的基因敲除技术是一种有效的下调靶基因的表达。这种技术通常用于单基因操作，但是它有局限性来检测基因之间的相互作用和共同作用。在这段视频的第一部分中，我们提出了两种策略，同时打掉两个基因的（称为双基因敲除）。我们表明这两种策略都能够有效地抑制这两个基因，卵黄蛋白原（VG）和ultraspiracle（USP），这是在调节反馈回路。这双基因敲除的方法可以用来分析基因之间的相互关系，可以很容易地应用在不同的昆虫种类。 该视频的第二部分是蜜蜂双基因敲除的治疗后长鼻扩展响应（PER）检测示范。市盈率法是一种标准的测试测量味觉感知的蜜蜂，这是一个重要的征兆，一个蜜蜂的行为成熟的速度有多快。大巢蜜蜂表示味觉感知行为发展的增加往往与花粉的觅食觅食专业化的发病年龄提前。此外，每次检测可用于识别代谢蜜蜂在饱食或饥饿状态。最后，结合配对不同的刺激气味，空调的蜜蜂也被广泛用于学习和记忆研究蜜蜂每次检测。

无论是单次注射，为期两天的注射策略明显减少VG（单因素方差分析，P &lt;0.001）（ 图2A）和USP（单因素方差分析，P &lt;0.001）（ 图2B）转录水平的蜜蜂6天之后的双链RNA注射。通过与双链RNA的混合物的单次注射和vg的第一和第二美国药典（vg的 / 美国药典 ）2天注射的美国药典转录抑制是显着低于两日的注射用美国药典 （ ?...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

在这个协议中，我们描述了两种策略，同时抑制这两个基因在蜜蜂（双基因敲除）。然后，我们提出了如何用长鼻延伸反应（PER）的检测，以研究双基因敲除对蜜蜂的味觉感知的效果。

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

rnai-mediated-double-gene-knockdown-gustatory-perception-measurement

Research

JoVE Journal

Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

14.8K 次观看.  Arizona State University. 在这个协议中，我们描述了两种策略，同时抑制这两个基因在蜜蜂（双基因敲除）。然后，我们提出了如何用长鼻延伸反应（PER）的检测，以研究双基因敲除对蜜蜂的味觉感知的效果。

视频: RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

使用光刺激神经元的负担得起的LED阵列

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

MPI Cyber​​Motion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

触觉空调和取样策略在蜜蜂触角的运动分析（<em&gt;蜜蜂</em&gt; L.）

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

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一种新的行为分析调查个人的味觉，可自由移动的熊蜂（

约束和自由飞行，蜜蜂的神经药理学操纵，

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

摘要

探索更多视频

此视频中的章节

双荧光原位杂交食脑切片

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

使用光刺激神经元的负担得起的LED阵列

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

MPI Cyber​​Motion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

触觉空调和取样策略在蜜蜂触角的运动分析（

嗅觉受体神经元在全山免疫标记

脑组织中的双管和同心微电极用于测量细胞外离子的信号

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（

约束和自由飞行，蜜蜂的神经药理学操纵，

MPI CyberMotion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分