我们感谢约阿希姆·埃伯向我们介绍的触觉学习模式，蜜蜂。这项工作是由群集支持的卓越277 CITEC，资助在德国卓越计划的框架。

1。准备蜜蜂<ol><li>花蜜或花粉觅食，被发现在该领域无论是从蔗糖进纸器或直接从蜂巢入口，而返回的觅食之旅。每一个单一的蜜蜂被捕获到的玻璃小瓶，用泡沫插件进入实验室作进一步处理，并立即采取关闭。 </li><li>在实验室中，捕获的蜜蜂进行了简要冷却冰箱在4°C不动，直到他们首先显示的迹象。 </li><li>每个单一的固定化蜜蜂被安装在一个小的金属管与粘合带之间的头部和胸部和腹部上。长鼻和天线是可自由移动的，应小心。 </li><li>油漆的固定用白色油漆的蜜蜂（ 例如无溶剂TIPP-EX），以堵塞视觉的复眼和单眼。 </li><li>加一小滴融化的蜡头后面的蜜蜂来解决它在录音磁带头部和胸部之间，以防止头部运动。 </li><li>一些在磁带上，更好地识别标记每个单蜜蜂和将管子固定的蜜蜂到一个潮湿的环境，以防止脱水。 </li><li>互相喂单一的蜜蜂，持续5秒的30％的蔗糖溶液，用注射器的液滴，并让所有蜜蜂恢复与触觉的调理协议开始之前30分钟。 </li></ol> 2。轻触式空调<ol><li>在调节之前，每个单个的蜜蜂将测试其长鼻扩展响应（PER）到30％蔗糖的刺激施加到的触角。由此，将吸管尖端具有交叉的虚拟线之间的打开的下颌骨。丢弃所有的蜜蜂不响应的PER蔗糖刺激。 </li><li>对于触觉空调使用的黄铜多维数据集（ 例如，3×5毫米）与平稳或刻图案， 例如 ，水平或垂直槽形成的光栅150微米波长，作为条件刺激（CS）。 F或非条件刺激（US）使用了30％的蔗糖溶液（户糖，在水中稀释）。 </li><li>黄铜立方体（CS）被放置到一个保持器上显微操作（ 例如 MM33Märzhäuser）的，以确保在调节过程中的精确定位。美国提出了填充用30％的蔗糖溶液，用注射器的蜜蜂。 </li><li>的调理过程包括五个审间5分钟的时间间隔（ITI）的触觉刺激（CS）和蔗糖溶液（US）的配对。将前面的显微操作器与所安装的触觉刺激（CS）中的一个单一的蜜蜂。位置CS缓慢，使得触觉刺激的表面是平行的蜜蜂的头部（ 图1中A和 B）。动物和触觉对象之间的距离应该是在测试蜜蜂触角的工作半径的范围内， 即在一个舒适的位置w的蜜蜂应该是能够扫描的触觉刺激第i个两个天线。在图1中所示的例子中，距离是3毫米。让蜜蜂扫描触觉刺激（CS），持续5秒。后的第3秒内，呈现出30％的蔗糖溶液（US）用注射器下长鼻液滴。使用的注射器的前端轻轻抬起长鼻。蔗糖刺激下的长鼻将引起每15个非条件。让蜜蜂舔蔗糖的奖励。使用秒表与一个报警信号，以维持精确的时间间隔期间空调。 </li><li>空调PER的用作蜜蜂学习成功的量度。蜜蜂开始后的第一个奖励配对，以应对CS演示文稿延长他们的长鼻，这表明他们期待即将出台的奖励。完全伸出长鼻过程中随时观察到3秒的时间窗口之前的触觉刺激呈现和蔗糖介绍，取得了积极的响应。无响应计数负面的。在OCC的PER的urrence必须注意由实验者。每次试验的比例绘制蜜蜂的市盈率在CS介绍。 </li></ol> 3。运动录音<ol><li>一个单一的驾驭蜜蜂触角变动记录有数字视频相机透镜（ 如巴斯勒A602f-2配备技术规格VZM 200的操作，在50 fps的通过一个防火电线连接）与一个合适的宏。是相机处于上方的动物中的自顶向下的视图（ 图1 A）。在图1中，在录音的空间分辨率为0.02毫米/像素。 </li><li>校准摄像头，通过记录一个10×10毫米的棋盘与从不同方位下的摄像头的目标的边缘的长度为1毫米的单的照片。校准可以用的摄像机标定工具箱Matlab的16。 </li><li>下面的相机镜头放置一个固定的蜜蜂。目前的触觉刺激，固定显微操作，给动物。继续以同样的方式所描述的触觉空调，和记录触角运动而蜜蜂被扫描对象。充分触角的工作范围和触觉的刺激，这是很重要的是可见的。刺激模式的选择，测试和，试验数字取决于实验的设计。 </li></ol> 4。数据分析<ol><li>在Matlab计算图像的背景。首先，随着时间的推移的平均灰度值，要对每一个像素计算。静态对象，如固定头的蜜蜂和触觉刺激，将构成图像的背景。移动的物体，如两个天线，不会成为背景的一部分。 </li><li>所记录的视频的每个帧的顺序加载，和当前帧的图像的背景之间的差异具有以被计算。相减的结果，强调的移动从帧到帧的图像中的那些部分。编号两坳一隆一斜坡，触角的蜜蜂的非零值（ 图1C）的唯一区域。 </li><li>进行进一步的处理，两个最大的非零象素值的区域被假定为与天线。要产生含有对每一个像素的值1，如果像素属于天线和值，否则为0，对于这两种天线的二进制掩码。这个面膜是为本土化触角提示后，以此为基础。要获得初步的掩模对整个图像的差分图像的各像素的灰度值进行比较，以一个预先定义的阈值。因为我们照顾的噪音阈值选择是相当低的。初步面膜之后，还进行两种类型的错误:第一，由于图像噪声小区域仍有部分的面膜。其次，属于天线的领域，可能不一定完全连接。后者多发生，如果背景具有大致相同的发光作为天线。为了消除这些文物，图像处理形态学操作应用， 即图像的侵蚀和扩张相结合，依赖于标准的Matlab的功能imerode和imdilate（见17页158-205作进一步的解释）。经过去噪的过程中，天线的假设仍然包含在相同的面具。因此，作为下一个步骤的二进制掩码集中不相交的区域，使用标准的Matlab:功能bwlabel（的图像分割算法的进一步详情，请参阅17页40-48）。 </li><li>在每个群集的像素的数目进行计数，并选择两个最大的群集。重心计算区分之间的左和右的天线（ 图1D）。 </li><li>群集的近端到远端的方向（参见图1D）中的最高值的像素在一个可以被定义为为每个天线的触角小费。 </li></ol>

<ol>
	<li>Page, R. E., Scheiner, R., Erber, J., Amdam, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+evolution+of+division+of+labor+and+foraging+specialization+in+a+social+insect+(Apis+mellifera++L.).">The development and evolution of division of labor and foraging specialization in a social insect (Apis mellifera L.).</a> Curr. Top. Dev. Biol. 74, 253-286 (2006).</li><li>Menzel, R., M&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+in+honeybees:+From+behavior+to+neural+substrates.">Learning and memory in honeybees: From behavior to neural substrates.</a> Annu. Rev. Neurosci. 19, 379-404 (1996).</li><li>Seeley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The wisdom of the hive. , (1995).</li><li>Staudacher, E., Gebhardt, M. J., D&#252;rr, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+and+mechanoreception:+neurobiology+of+active+tactile+sensors.">Antennal movements and mechanoreception: neurobiology of active tactile sensors.</a> Adv. Insect Physiol. 32, 49-205 (2005).</li><li>Martin, H., Lindauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinnesphysiologische+Leistungen+beim+Wabenbau+der+Honigbiene.">Sinnesphysiologische Leistungen beim Wabenbau der Honigbiene.</a> Z. Vergl. Physiol. 53, 372-404 (1966).</li><li>Kevan, P. G., Menzel, R., Mercer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Texture+sensitivity+in+the+life+of+honeybees.">Texture sensitivity in the life of honeybees.</a> Neurobiology and behavior of honeybees. , 96-101 (1987).</li><li>von Frisch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tanzsprache und Orientierung der Bienen. , (1965).</li><li>Waser, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flower+constancy:+definition,+cause,+and+ceasurement.">Flower constancy: definition, cause, and ceasurement.</a> Am. Nat. 127, 593-603 (1986).</li><li>Kevan, P. G., Lane, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flower+petal+microtexture+is+a+tactile+cue+for+bees.">Flower petal microtexture is a tactile cue for bees.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 4750-4752 (1985).</li><li>Erber, J., Kierzek, S., Sander, E., Grandy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+in+the+honeybee.">Tactile learning in the honeybee.</a> J. Comp. Physiol. A. 183, 737-744 (1998).</li><li>Kisch, J., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+conditioning+of+antennal+movements+in+the+honey+bee.">Operant conditioning of antennal movements in the honey bee.</a> Behav. Brain. Res. 99, 93-102 (1999).</li><li>Scheiner, R., Erber, J., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+and+the+individual+evaluation+of+the+reward+in+honey+bees+(Apis+mellifera+L.).">Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. 185, 1-10 (1999).</li><li>Erber, J., Scheiner, R., Adelman, G., Smith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+learning.">Honeybee learning.</a> Encyclopedia of Neuroscience. , (2004).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Sch&#228;fer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera).">Classical conditioning of proboscis extension in honeybees (Apis mellifera).</a> J. Comp. Psychol. 97, 107-119 (1983).</li><li>Dacher, M., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+NO-synthase+and+nicotinic+receptors+in+learning+in+the+honey+bee.">Involvement of NO-synthase and nicotinic receptors in learning in the honey bee.</a> Physiol. Behav. 95, 200-207 (2008).</li><li>Haralick, R. M., Shapiro, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Computer and Robot Vision. I, (1992).</li><li>Erber, J., Galizia, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+antennal+learning+in+the+honey+bee.">Tactile antennal learning in the honey bee.</a> Honeybee Neurobiology and Behavior. , 439-455 (2012).</li><li>Pankiw, T., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pheromones,+hormones,+and+handling+on+sucrose+response+thresholds+of+honey+bees+(Apis+mellifera+L.).">Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. 189, 675-684 (2003).</li><li>Harris, J. W., Woodring, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+stress,+age,+season+and+source+colony+on+levels+of+octopamine,+dopamine+and+serotonine+in+the+honey+bee+(Apis+mellifera+L.)+brain.">Effects of stress, age, season and source colony on levels of octopamine, dopamine and serotonine in the honey bee (Apis mellifera L.) brain.</a> J. Insect. Physiol. 38, 29-35 (1992).</li><li>Scheiner, R., Kuritz-Kaiser, A., Menzel, R., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+responsiveness+and+the+effects+of+equal+subjective+rewards+on+tactile+learning+and+memory+of+honeybees.">Sensory responsiveness and the effects of equal subjective rewards on tactile learning and memory of honeybees.</a> Learn. Mem. 12, 626-635 (2005).</li><li>Mujagic, S., Sarkander, J., Erber, B., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance+and+different+forms+of+associative+learning+of+the+honey+bee+(Apis+mellifera+L.)+in+the+field+and+laboratory.">Sucrose acceptance and different forms of associative learning of the honey bee (Apis mellifera L.) in the field and laboratory.</a> Front. Behav. Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. A. 195, 325-339 (2009).</li><li>Friedrich, A., Thomas, U., M&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+at+different+satiation+levels+reveals+parallel+functions+for+the+cAMP-protein+kinase+A+cascade+in+formation+of+long-term+memory.">Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory.</a> J. Neurosci. 24, 4460-4468 (2004).</li><li>Erber, J., Pribbenow, B., Grandy, K., Kierzek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+motor+learning+in+the+antennal+system+of+the+honeybee+(Apis+mellifera+L.).">Tactile motor learning in the antennal system of the honeybee (Apis mellifera L.).</a> J. Comp. Physiol. A. 181, 355-365 (1997).</li><li>Lambin, M., D&#233;glise, P., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+as+indicators+of+odor+detection+by+worker+honeybees.">Antennal movements as indicators of odor detection by worker honeybees.</a> Apidologie. 36, 119-126 (2005).</li></ol>

没有利益冲突的声明。

蜜蜂制备:收集和固定的蜜蜂应该迅速完成，以保持动物的低应力水平。应激反应的影响，因此，可能有间接影响的学习表现在蜜蜂19，20。的应力水平可以降低由放置玻璃小瓶，与直接在冰上的蜜蜂采集后立即迅速固定它们。必须加以考虑，蜜蜂需要更多的时间来恢复麻醉期间之前的时间越长。 视觉触觉学习有负面影响，在蜜蜂18。因此，重要的...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>花粉的蜜蜂觅食，L. </td><td></td><td></td><td>私人荨麻疹</td></tr><tr><td>玻璃小瓶中（22×45毫米） </td><td>卡尔·罗斯GMBH＆CO </td><td> X655.1 </td><td></td></tr><tr><td>铜管的，10×30毫米</td><td></td><td></td><td>自产</td></tr><tr><td>铜立方体，3 x 5毫米</td><td></td><td></td><td>自产</td></tr><tr><td> TIPP-EX（无溶剂） </td><td> TIPP-EX GMBH＆CO KG </td><td></td><td></td></tr><tr><td>蔗糖溶液（30％） </td><td></td><td></td><td>家居糖用水稀释</td></tr><tr><td>乙醇（70％） </td><td>卡尔·罗斯GMBH＆CO </td><td> 9065.3 &lt;/ TD&gt; <td></td></tr><tr><td>注射器（1ml）和针（0.6×25） </td><td>卡尔·罗斯GMBH＆CO </td><td> 59.1 </td><td></td></tr><tr><td>秒表</td><td>卡尔·罗斯GMBH＆CO </td><td> L423.1 </td><td></td></tr><tr><td>微操作机器人</td><td> MärzhäuserMM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td>数码摄像机</td><td>巴斯勒A602f-2 </td><td></td><td></td></tr><tr><td>微距镜头相机</td><td>技术规格VZM 200 </td><td></td><td></td></tr><tr><td> MATLAB R2009b </td><td> The MathWorks公司</td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

蜜蜂（ 蜜蜂 L.）是典型的社会性昆虫，其复杂的劳动分工和联想学习能力1，2众所周知的。工蜂花上半年在昏暗的蜂巢，他们的生活，他们是护士的幼虫或建设正六边形梳子食品（ 如花粉或花蜜）和育雏3。非凡的多感官触角，触角和各种触觉介导的任务，包括蜂巢5号楼和模式识别6中发挥了举足轻重的作用。以后在生活中，每个单一蜜蜂离开蜂巢饲料，用于食品。然后，一只蜜蜂学习歧视有利可图的食物来源，记住它们的位置，并将其传达到它的巢队友7。蜜蜂用不同的花卉信号，如颜色或气味，7，8，但也有触觉线索，从花瓣表面形成多感官记忆的食物来源。在实验室的合作的条件，蜜蜂可以训练，学习模式食欲，区分触觉的物体的特征，如边缘或沟槽，其触角10日，11日，12日，13。这种学习模式是密切相关的经典嗅觉空调的长鼻延伸反应（PER）的利用，蜜蜂14。在实验室中的触觉学习范式的优点是将与各种生理测量，包括触角的运动模式的分析，对学习行为实验相结合的可能性。

在下面的实验中，我们研究了，触觉学习如何在蜜蜂的行为影响触角采样。对于这一点，我们监测的天真和空调蜜蜂触角尖端之前和期间提交的触觉刺激的运动。 首先，自发触角花粉觅食一组（N = 42）的运动记录，持续1分钟。蜜蜂的二分之一（N = 21），然后，空调的配对5次用30％的蔗糖的奖励的触觉刺激。这是空调基。触觉刺激是30×50毫米的黄铜表面的立方体，一个刻有的?...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

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

在这个协议中，我们展示了如何驾驭的条件蜜蜂的触觉刺激和引进的2D动作捕捉技术的运动学分析精细尺度的触角采样模式。

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 ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

10.5K 次观看.  Bielefeld University. 在这个协议中，我们展示了如何驾驭的条件蜜蜂的触觉刺激和引进的2D动作捕捉技术的运动学分析精细尺度的触角采样模式。

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

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 ...

科研

神经科学

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

环境科学

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

木蚁视觉经典调理

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

行为学

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

生物学

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). ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

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 ...

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 ...

触觉空调和取样策略在蜜蜂触角的运动分析（

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