This project was funded by ARC grant DP0986021 and NHMRC grant 585442. ABB is supported by an ARC Future Fellowship (FT140100452). JAP is supported by an iMQRES scholarship awarded by Macquarie University and by a DAAD-Doktorandenstipendium awarded by the German Academic Exchange Service. JMD is supported by CNRS and University Paul Sabatier.

1.药品监督管理局铁甲蜜蜂<ol><li> 口服药物治疗 <ol><li>通过混合257克蔗糖用​​500毫升水制备1.5M的蔗糖溶液（它是比较容易溶解于沸水蔗糖的这个量）。储存在4℃直至使用蔗糖溶液。 注:蔗糖溶液中某些微生物提供了一个非常好客的环境，因此很容易被污染和令人不快的蜜蜂。散装蔗糖溶液可以等分并储存在-20℃直到使用。 </li><li>决定适当的药物剂量（如何实现的，在下面的讨论部分被寻址），并且制备溶液，使得优选的药物剂量溶解在20μL的蔗糖溶液（ 例如 ，以提供20微克，在稀释药物的1毫克/毫升的比例）。根据Felsenberg等线束蜜蜂。 （2011年）16。做到这一步，至少12小时的药物治疗前，以确保蜜蜂不再强调Ø从提出药物溶液时，利用UT斯达康。 注:对于更一致的结果，最好是饿死的蜜蜂（通过在34°C和70％的湿度将驾驭蜜蜂在孵化器）O / N。 </li><li>用微量，触摸的1.5蔗糖一滴水到驾驭蜜蜂的天线。当长鼻延伸，触及的直接含有药物对蜜蜂的长鼻1.5蔗糖20微升滴。确保蜜蜂消耗的一切。由于车辆控制使用1.5米不添加药物的蔗糖溶液。 注:蔗糖溶液的量可能需要根据实验计划进行调整。如果食欲调节的目的，只是之前的训练喂养蜜蜂将与蜜蜂的响应能力。 </li><li>丢弃或搁置不消耗所有的蔗糖蜜蜂。 注意:如果大量蜜蜂失败饮蔗糖溶液，馈送时间表可能需要进行调整。 </li></ol></li><li> 注射到胸腔 <ol><li>在蜜蜂准备药物林格17如下: <ol><li>混合和高压釜7.45克氯化钠，0.448克氯化钾，0.812克的MgCl 2，0.735克CaCl 2，54.72克蔗糖，4.95克D-葡萄糖，并在1,000ml水中2.48克HEPES。存储铃声时，因为它容易被污染要小心。分装和储存在林格-20°C，直到使用。 </li><li>溶解在林格氏溶液中的药物，然后稀释以使所需量存在于5微升。作为一个例子，如果蜜蜂与药物的5微克进行处理，将1克最初可以溶解在1ml中林格氏，稀释前1:1000在林格氏为1微克/微升的最终溶液。 注意:可替换地，可商购的PBS（磷酸盐缓冲的萨兰）可以用来代替林格溶液。 </li></ol></li><li>您可以透过掰双刃刀片与角落microscalpel一个刀片夹持器。安装刀片片段刀架，以便它使一个很好的刀片用锋利的终点。 </li><li>在立体显微镜，小心使用microscalpel削减2毫米的孔就在上面的盾，旁边一个蜜蜂的胸部的后翼过程。避免切割太深，因为这可能会伤害飞行肌，并注意避免机翼铰链。理想的情况下，只切三边，以使角质层的襟翼以后可以向后折迭以封闭损伤部位。 </li><li>使用含有在胸廓孔顶部的药物微量，存款5μlL林格（或PBS）中。显微镜下仔细监测以确保整个滴被吸收到血淋巴。使用林格氏（或PBS），其为车辆的控制。 </li><li>如果可能的话，将角质层皮瓣回过孔。 5-10小时后，将重新连接和密封。 注意:作为替代该技术中，使用玻璃注射器，微升直接注射1到胸腔打开SMA后在系带（在盾片的后部区域的横向线）的中间LL孔用注射器针（直径:0.6毫米，G:23）。这就避免了需要首先切断胸部用解剖刀和注射部位较小，但这种方法会留下暴露注射部位。 </li></ol></li><li> 单眼注入 注意:这是适用于整个头壳传递分子，进入血淋巴的方法。 <ol><li>制备药物如1.2.1，但调整药物浓度，使得所要求的剂量将被包含在1微升林格氏或PBS（体积较少，可以通过单眼孔比通过胸廓吸收）。 </li><li>准备一个microscalpel为1.2.2。在立体显微镜，通过填充蜡颈部缝隙锁定驾驭蜂的头部到位。为了避免损坏触角嗅觉受体或其它细胞，可能是我使用的低温熔融的蜡（ 例如，牙科蜡）mportant评估行为（ 例如，嗅觉学习）。然后仔细镜头下插入microscalpel的尖去掉中间单眼镜头，轻轻地摆脱头壳免费的镜头。 注意:也可以小心地将蜡在天线以防止移动。 </li><li>认真吸取药到单眼洞。等到一切都考虑到头壳。从天线清除牙蜡和允许蜂继续的实验程序之前休息一会儿。使用林格氏（或PBS），其为车辆的控制。 </li></ol></li><li> 注入单眼道 注:单眼道中含有大量的纤维，连接到中央大脑18的大部分地区。这种处理方法使得能够施加化合物仅脑，但不是针对大脑的特定子区域。 <ol><li>蜜蜂准备在1.3.2。并取出位单眼镜头与T一个microscalpel的IP为1.3.3。 注意:此可注射之前进行长达2小时。根据我们的经验，喂养蜜蜂能够更好地应付这种手术比饥饿蜜蜂</li><li>填充配小计10微升玻璃注射器（ 例如，33，直径210微米）针药物溶液1.3.1准备。 </li><li>使用手动显微通过单眼视网膜插入注射器尖端进入头壳到50μm的深度和注入250 NL的溶液。 </li><li>使用后，冲洗注射器3次用蒸馏水，然后用75％乙醇3次。 </li></ol></li><li> 显微注射到特定的大脑结构 注意:除了上面提到的系统的处理，能够进行微量注射到特定的大脑结构。这允许一个或多个脑区域中的药理学处理，而留下其他不受影响。这工作最好的脑区，很容易由前脑表面（ 如触角叶，蘑菇体花萼或垂直波瓣，或视叶）承认，但其他地区都有针对性。请注意，取向（前/后，背/腹）指的是体轴，而比对神经轴19。 <ol><li>准备在林格氏或PBS的药物中的相同方式在1.2.1，加入荧光（ 例如，0.5毫克/毫升的葡聚糖，Alexa的546或568氟）或非荧光染料（ 例如 ，1毫亚甲蓝）。 注:加入的荧光染料的将允许注射位置的验证后，在实验结束（使用共焦显微镜，以下脑解剖），而非荧光染料允许在实验过程中直接监测。 </li><li>为了使注射玻璃吸管，插入正确的直径的玻璃毛细管到电极拉马的持有人夹（1.0毫米的标准保持呃包含在材料清单中提到的微量）。调整拉热设置，以产生一个大约为0.5厘米长的端头（设置将是每牵拉不同，即使使用相同的模型）。 注:在理想情况下，这两个移液器从一个玻璃拉应具有相同的长度和形状，这样既可以用。 </li><li>下一个立体显微镜，打破提示，以获得约10-15微米的外径，基于使用上插入眼一个刻度的比例目测。刻度上的步骤是由制造商定义的，并且可以对所使用的放大率进行校正。 </li><li>然后，填充玻璃吸管与溶液注入。如果使用具有长丝玻璃毛细管，通过将背面侧入药液充满吸管，使用微加载提示否则填充小费。 </li><li>插入填充玻璃吸管到微量的毛细管保持器，它是由一个手动或电子英里控制cromanipulator。 </li><li>校准微量注入所需体积（0.5-2 NL，这取决于大脑结构的定位的大小）。对于这一点，直接注射到含有矿物油中的小的培养皿，并测量与刻度液滴的直径。直到达到所需音量更改设置。 </li><li>用柔软的牙模为1.3.2，切开口进入头壳的前部，使用前microscalpel修复利用，蜜蜂的头，用三个切口:一个略低于平均单眼（腹），一个在的天线的上方右或左眼和一个边界茎（背侧）。用一块牙科蜡保持打开挡板到位。 </li><li>小心推腺体和气管躺在大脑顶部预留使用细镊子，然后做一个小破入神经鞘（非常薄的大脑周围膜），有针对性的大脑结构之上。 注:如果许多蜜蜂在一次治疗，此过程可以提早进行;但是，要小心不要离开在这种状态太长（不超过30分钟）蜜蜂，因为他们的大脑可能变干。 </li><li>将尖端插入所需的大脑区域，并调整深度垂直于脑表面（ 例如，60微米的蘑菇体肾盏）。注射预设量。对于横向的大脑区域，双边注入（ 即做一注入每个半球）。如果使用非荧光染料，确保在注射而注射后观察发生在右侧区域。如果使用荧光染料使用用荧光观察系统立体显微镜做在荧光灯下是相同的。 </li><li>之后，将开瓣又回到了蜜蜂的头。熔融二十烷的结晶，其直径大约为1毫米，用细线围绕微烙铁的前端缠绕（熔融温度为35-37℃）和密封该切口。这将大大降低死亡率。 </li><li>释放从线束行为分析蜂（但见讨论），或保留在线束上的内敛蜜蜂的实验- 例如，长鼻扩展反射（PER）测试20。 </li><li>如果使用荧光染料，确保喷射击中感兴趣区域之后的实验是在使用共聚焦激光扫描显微镜（ 图1）。 注意:指定更深脑区域（其中，这将是很难看到在注射阶段非荧光染料）时，这是特别有用的。 </li></ol></li></ol> 2.药品监督管理局方法自由飞行的蜜蜂<ol><li> 口服药物治疗 <ol><li>以同样的方式制备药物如步骤1.1.1-1.1.2。添加药液至馈线和在冰箱中放置用于存储。 注意:任何馈线会做，如倒置瓶盖或倒在纸巾一个罐子。 </li><li>训练蜜蜂含有1M或0.5重力给料机通过将接近蜂巢馈线蔗糖的解决方案。一旦蜜蜂开始在料器觅食，逐渐它，直到它是在一个舒适的距离，以免被蜇（最少5米）移动更远。 </li><li>为了画图标志蜜蜂来跟踪单个的蜜蜂。使将要使用的所有的颜色组合的列表。当一只蜜蜂在土地馈线，仔细标记与两种颜色腹部，使该组合该名单上的音符。 </li><li>交换自流式给料为含有药物/蔗糖溶液饲养。记标记的蜜蜂是参观馈线。捕捉任何无人盯防的蜜蜂逛进纸器蜜蜂是多产的招聘人员，和蜜蜂参观迷药进纸器可以迅速失控的数字。这是特别有问题的，如果相同的实验是要在连续天进行，如幼稚蜜蜂可能不再天真。 注意:作为替代训练个体蜜蜂馈线，以前提交■找成功送入药里掺蔗糖水将整个蜂巢21 - 23。 </li></ol></li><li> 局部治疗 注意:目的是溶解所关注的化合物在可穿透蜡质昆虫角质层的溶剂。不同的溶剂可以用于此目的。最常用的包括丙酮，二甲基甲酰胺（DMF）和二甲亚砜（DMSO）中。 <ol><li>评估其溶剂最适合手头的化合物。如果一个强大的表型是由溶解在每一个不同的溶剂，小心过量（如瘫痪或死亡），治疗蜜蜂（步骤2.2.2），用高剂量（ 例如 20微克可卡因7）预计监测时间，直至瘫痪或死亡。 </li><li>使用1微升微毛细管（或微量，其可以安装在一个适当的重复分配器）和微毛细管保持器，吸取药液的1微升（ 例如，381;可卡因克/微升）到毛细管。驱逐下降，精心绘制它到一个显着蜜蜂的胸部。覆盖大面积的可能的解决方案，而不是留下了坚实的下降，因为蜜蜂是那么容易梳理其关闭。小心不要让该化合物接触翼铰链，或本可以得出其关闭胸腔，沿着机翼在那里将蒸发而不被吸收进入血淋巴。 注意:根据不同的研究的目的，这种方法也可用于施用药物蜜蜂的腹部。然而，当施加到胸部24。这种方法同样适用与利用，与自由飞行蜜蜂药物到达CNS的更快和数量较多。 </li></ol></li><li> 挥发处理 <ol><li>溶解药物（以前这种方法已被用于递送可卡因蜜蜂10）在100％的乙醇。为了确保溶解性，不使用盐酸盐或其他盐形式的药物如果可能的话。当进行稀释制备它，这样的量被传递到蜂是存在于100微升。使用纯乙醇作为车辆控制。 </li><li>为了创建一个灯丝，使用相同的步骤麦克朗和赫希25。 <ol><li>简要说明:紧紧卷起镍铬丝周围钉子和附加到两个电导线（一个在灯丝的两端）。取出钉子。剩余的镍铬合金线圈被称为灯丝。 </li><li>线程通过精心钻孔两条电线中的50ml离心管中，这应该是所选择的温度抗性的盖子。胶丝与液态硅的地方。 注意:这将使得管密闭。这是必要的，以避免二次暴露于实验者并确保蜜蜂用适当的剂量进行处理。 </li></ol></li><li>附着导致灯丝到电源的导线。使用热电偶测量的温度灯丝，试验用不同的电压/电流的组合，直到一个其导致在适当的温度分布为药物在问题，理想的是，一个允许用于加热或更少的10秒。这是非常重要的，请参阅相关文献（ 例如，为了可卡因挥发需要将其加热到至少200℃，但在温度超过350℃时分解成次级化合物26）。 </li><li>仔细移液器100微升含乙醇溶液到灯丝的药物。散布在液体以上之多长丝表面尽可能这将增加蒸发效率。留在室温下暴露在灯丝直到所有的乙醇蒸发。 注意:如果乙醇不能充分地蒸发，蜜蜂将与选择和乙醇两者的药物进行治疗。蜜蜂对酒精非常敏感的，有些药物有乙醇协同作用，这将偏置前perimental结果。 </li><li>一旦乙醇完全蒸发（药物的沉淀通常可以在显微镜下干长丝看到），抓在50ml管中的自由飞行蜂。小心关闭含丝盖子。 </li><li>打开电源10秒，关闭电源，等待另一个50秒（以使挥发的化合物冷却，从而凝结或存款）。释放蜜蜂。 注:虽然这种治疗方法效果出色的自由飞行的蜜蜂，它可以是一样有效地利用，蜜蜂使用。简单地附加50ml管内的驾驭蜂。蜜蜂之间2.3.4描述刷新灯丝。对于更高的吞吐量，多个细丝可以并行地使用。 </li></ol></li></ol>

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The authors have nothing to disclose.

上面提到的方法允许任何自由飞行或利用，蜜蜂的简单，有效和有力的治疗方法。这些方法有许多实验范式和生物学问题（ 表1）兼容。所有的自由飞行的方法可以很容易地应用到利用，蜜蜂。相反的是不太成功的，但是，由于暂时克制和侵入性治疗方法往往能危及蜜蜂的飞行能力。 该方法已经从大脑为中心的立体呈现。这不是由于技术固有的局限性，但作者的?...

由于卡尔·冯·弗里希阐明他们的舞蹈语言1，蜜蜂仍然是一个热门的研究物种的动物行为和神经生物学的研究人员。近年来新学科无数已经出现在这两个领域，和其他几个学科（如分子生物学，基因组学和计算机科学）已经出现和他们一起的交集。这导致了新的理论和模式的快速发展，为理解的行为从活动结果如何神经系统之内。由于独特的生活方式，丰富的行为库，并且易于实验和药理操纵的，蜜蜂一直维持在这个革命的最前沿。 蜜蜂被用来研究基本神经生物学问题那些潜在学习和记忆2,3，决策4，嗅觉5，或可视处理6如。在最近几年，议员EY蜂甚至被用作研究主题通常保留用于医学研究，例如成瘾药物7的影响的模型- 11，睡眠12，老化13，或机制基础麻醉14。 对于不同的经典遗传模式生物（ 例如 ， 果蝇 ， 线虫 ，M.家鼠 ），也有极少数可用于蜜蜂操纵的神经功能的遗传工具，虽然这是目前改变15。相反，蜜蜂的研究主要依靠药理操作。这是非常成功;然而，蜂研究的多样性是使得所需的药理给药方法的范围内。研究与蜜蜂满足高度多样化的问题，是由来自不同学科和背景的研究人员研究，并采用了多种实验方法。许多RESE弓问题需要蜜蜂要么是自由飞行，在他们的殖民地，或两者相互作用的自由。这可以使它很难跟踪个别实验动物，并使得限制或插管不可行的。 容纳蜜蜂研究的多样性，需要多种药物递送方法，允许强大和灵活的管理，同时确保药物动力学和药效型材，该方法的侵袭，其可靠性，适合所讨论的范例。由于这些多样化的需求，大部分的研究小组已经开发出了自己独特的给药方法。到目前为止，这一直是蜜蜂研究界的力量;它导致了允许在不同的情况下的同种药物的给药方法阵列的发展。我们的目标不是要开发蜜蜂药理操纵一个标准化的方法，而是突出的方法已被证明是特别成功的，并帮助研究人员采用这些。我们讨论的是如何工作的基本原则，以及它们的优点和缺点。

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>Any supplier will do</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333&amp;nbsp;</td><td></td></tr><tr><td>Magnesium Chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Calcium Chloride dihydrate</td><td>Sigma-Aldrich</td><td>C8106</td><td></td></tr><tr><td>Dextrose monohydrate</td><td>Sigma-Aldrich</td><td>49159</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Protection Wax</td><td>Dentaurum</td><td>124-305-00</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>dimethylformamide</td><td>Sigma-Aldrich</td><td>D4551</td><td></td></tr><tr><td>95% Ethanol</td><td>Sigma-Aldrich</td><td>493511</td><td></td></tr><tr><td>Glass capillary</td><td>WPI</td><td>1B100F-3</td><td></td></tr><tr><td>23 G NanoFil needle</td><td>WPI</td><td>NF33BV-2</td><td></td></tr><tr><td>Very fine forsceps</td><td>Dumont</td><td>0208-55-PO</td><td></td></tr><tr><td>Electrode puller</td><td>SRI</td><td>2001</td><td></td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.01</td><td></td></tr><tr><td>Eicosane</td><td>Sigma-Aldrich</td><td>219274</td><td></td></tr><tr><td>manual micromanipulator</td><td>Brinkmann Instrumentenbau</td><td>MM-33</td><td></td></tr><tr><td>electronic micromanipulator</td><td>Luigs &amp;amp; Neumann Feinmechanik + Elektortechnik</td><td>Junior unit XYZ</td><td></td></tr><tr><td>stereomicroscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>soldering iron</td><td>Weller</td><td>WESD51</td><td></td></tr><tr><td>Dextran, Alexa Fluor 546, 10,000 MW</td><td>ThermoFisher Scientific</td><td>D-22911</td><td></td></tr><tr><td>Dextran, Alexa Fluor 568, 10,000 MW</td><td>ThermoFisher Scientific</td><td>D-22912</td><td></td></tr><tr><td>small Petri dish</td><td>Sigma-Aldrich</td><td>P5481</td><td></td></tr><tr><td>mineral oil</td><td>Sigma-Aldrich</td><td>M5904</td><td></td></tr><tr><td>50 ml Centrifuge tube</td><td>ThermoFisher Scientific</td><td>339652</td><td></td></tr><tr><td>forceps</td><td>Australian Entomological Supplies</td><td></td><td></td></tr><tr><td>Blade holder and breaker</td><td>Australian Entomological Supplies</td><td>E130</td><td></td></tr><tr><td>Feather double edged razor blade</td><td>ThermoFisher Scientific</td><td>50-949-135</td><td></td></tr><tr><td>Nichrome wire</td><td></td><td></td><td>Any supplier will do</td></tr><tr><td>Electrical wires</td><td></td><td></td><td>Any supplier will do</td></tr><tr><td>Model paint</td><td>Tamiya USA</td><td>Depends on colour</td><td></td></tr><tr><td>Repeating dispenser</td><td>Hamilton company</td><td>PB-600-1</td><td></td></tr><tr><td>Glass syringe</td><td>WPI</td><td>NANOFIL</td><td></td></tr><tr><td>flourescence viewing system</td><td>Nightsea</td><td>SFR-GR</td><td></td></tr><tr><td>graticule</td><td>ProSciTech</td><td>S8014-24</td><td></td></tr><tr><td>microcapillary with holder</td><td>Drummond</td><td>1-000-0010</td><td></td></tr><tr><td>Liquid silicone</td><td></td><td></td><td>Any supplier will do</td></tr><tr><td>Thermocouple</td><td>Digitech</td><td>QM-1324</td><td></td></tr><tr><td>Micropipette</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

neuropharmacological manipulation of restrained and free-flying honey bees, apis mellifera

蜜蜂展示惊人的学习能力和先进的社会行为和沟通。此外，他们的大脑体积小，便于可视化和学习。因此，蜜蜂也早已之间神经生物学家和neuroethologists为研究社会和自然行为的神经基础青睐的机型。是很重要的，但是，用于研究蜜蜂不与行为干扰的实验技术正在研究中。由于这个原因，一直需要开发出一系列的为蜜蜂的药理学处理的技术。在本文中，我们演示了具有广泛的药理剂治疗内敛或自由飞行的蜜蜂的方法。这些包括非侵入性的方法，如口服和局部治疗，以及更侵入性的方法，其允许在任一全身或局部的方式精确药物递送。最后，我们讨论每种方法的优点和缺点，并描述常见的障碍以及如何最好地克服它们。我们的结论与适应实验方法的生物学问题，而不是周围的其他方式的重要性进行了讨论。

用于上述方法的代表性结果的选择示，主要是为了证明方法允许药剂到达脑部，并影响蜜蜂行为。 对大脑处理具体影响如下胸部注射容易获得。 因为药剂注入通过胸廓可作用于在主体多个目标，并在到达大脑之前得到稀释到体内，这种技术可以提高可能特异性的担忧。尽管如此，它...

Watch this Scientific Journal Video about Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera at JoVE.com

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

这份手稿介绍了管理药剂对蜜蜂，包括自由飞行的蜜蜂简单的无创方法，以及更具侵入性的变异，让内敛的蜜蜂精确的局部治疗多种协议。

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 ...

neuropharmacological-manipulation-restrained-free-flying-honey-bees

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

9.9K 次观看.  Volda University College. 这份手稿介绍了管理药剂对蜜蜂，包括自由飞行的蜜蜂简单的无创方法，以及更具侵入性的变异，让内敛的蜜蜂精确的局部治疗多种协议。 

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

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

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

蜜蜂匀浆中磷脂酶 C 活性的检测

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

生物化学

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

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 ...

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

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

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

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

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 ...

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). ...

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 ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

SwarmSight: 利用共同的准备和常规硬件实时跟踪昆虫的天线运动和喙的延伸反射

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

约束和自由飞行，蜜蜂的神经药理学操纵，

摘要

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此视频中的章节

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

蜜蜂匀浆中磷脂酶 C 活性的检测

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

触觉空调和取样策略在蜜蜂触角的运动分析（

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

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

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

SwarmSight: 利用共同的准备和常规硬件实时跟踪昆虫的天线运动和喙的延伸反射