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，但作者的个人利益，而是因为。没有理由为什么这些方法不能用于研究其他器官。然而，可能需要小的修改，以使该方法更适合于其它器官系统。例如，虽然局部治疗意欲到达脑部，通常施加到胸部，它可能是更好的应用THIs至腹部如果预定目标是卵巢。类似地，注射剂可以很容易地应用于除了胸部或头部其他区域（ 例如，腹部器官可通过腹部骨片之间注入定位）。 其中的化合物可以给予蜜蜂而言，实在是没有限制。通常情况下，人们服用药物化合物，如信号分子21或他们的拮抗剂32和定制肽28。然而，一直在考虑应用的问题，如农药33和人为污染物34蜜蜂化合物给药最近有所增加。最近，施用的化合物已开始包括与基因表达的直接干扰RNA分子，如双链RNA激活RNA干扰途径35或甚至微小RNA 36和拮抗37。并不是所有的方法同样适合所有化合物。这也许是最好的苦味或酸味的化合物，使白糖水难吃的蜜蜂所示，从而防止它们消耗它。易碎分子，如RNA或某些多肽，当挥发过程中加热或放置在如DMF严酷溶剂被分解。理解的是施用什么，以确保它生存的治疗过程中的化学是非常重要的。 获取一个药剂变成蜜蜂是容易的部分，但也有应在执行药理实验时，绝不能掉以轻心三大担忧。第一是找出有问题的实验良好的剂量。取决于药物，有可能已被公开可获得的文献，但是在大多数情况下，这将必须通过文献检索，知情猜测，并且剂量反应曲线的混合物来解决。根据实验方案的复杂程度，这可能是有用首先产生在一个更简单的生物测定的剂量-响应曲线（ 例如，量化的整体运动或存活），以获得一剂量范围值得尝试在一个更复杂的生物测定的更好的主意。在我们的实验室中，起始剂量在蜂文献或通过执行基于来自啮齿动物文献数据的毫克/公斤转换要么找到。从这个起点，蜜蜂与起始剂量，加2或3个剂量的10倍起始剂量（较大和较小的处理例如，如果起始剂量为1毫克，0.01，0.1，10，和100毫克的也将是使用），当然还有一个适当的车辆控制。 第二个问题是稍微挑剔:药物特异性。大多数药物都不能与蜜蜂，或任何其他昆虫开发，记在心里。正因为如此，脱靶效应是常见的（ 例如，米安色林，脊椎动物血清素受体拮抗剂38，被长期被认为是一种昆虫章鱼胺能受体拮抗剂，但最近的网络连接ndings表明蜜蜂它也是一个多巴胺受体拮抗剂39）。对该问题的一个普通的解决办法是，而不是依赖于只是一种药物，具有一套已知具有在共同感兴趣的靶的药物，以重复相同的实验。基本上，如果几种药物是公知的阻断某一目标，观察在不同的药物相似的结果应该给予更大的信心，该药物具有预期的效果，因为不同的药物通常具有独特的脱靶轮廓。 最后一个问题涉及确保药物作用的地方应该是演戏。在这方面，总是会有特异性和侵袭之间的折衷。系统的治疗方法一般在至少是侵入性的，但没有在那里在蜂体药物是具有其效果的控制。即使对于目标组织的显微注射的药物可以与血淋巴的蜂身体的其他部分移动。这个问题是怎么解决的东东DS由提出的问题的通知。对于某些实验，解剖位置是无关紧要的，而对于其他人，这是重要的唯一的问题。解决这一问题的最好方法是，开始与全身治疗，逐渐通过使用越来越多的具体方法缩小到解剖位置。如果所研究的行为与侵入性治疗方法中特别不相容，这可能是值得尝试做了一系列的实验以非常具体的药物治疗之前，它解构成更简单的元件。 药物渗漏的这个问题，即使有自由飞行的蜜蜂，其中药物可影响非目标蜜蜂口服治疗比较夸张。觅食的蜜蜂采集花蜜在外地带回他们的殖民地。他们将在返回而不是吸收它卸载大部分在蜂巢的蔗糖溶液。在蜂巢它是装在细胞中，脱水，并作为蜜存储。因为对此，药物可能会影响非目标蜜蜂。更具体的方法（如显微注射）此问题最小化。 有了这些告诫铭记，并处理得当，蜜蜂的神经药理学操作可以是一个非常强大的工具。而为蜜蜂15正在开发，因为它们的社会生活的转基因的工具，这是不可能的，转基因将永远是一种简单且可靠的方式进行这些类型的实验。因此，可能的是药理学将继续在未来蜂研究的一个重要因素。虽然一些研究人员蜂取得了标准化的实验方法调用40，在这种情况下，这将是一个错误。蜜蜂系统的电源部分一直是实验方法的多样性，以及如何技术已经开发出心中真正的生物学问题，而不是周围的其他方法。我们确保它仍然是重要的手头问题的最适当的方法的使用。如果对比以前的研究是关键，标准化的协议必须严格遵守。然而，利用建立的协议采用标准化的方法的缘故绝不允许在可以打开新的实验可能性的新方法的发展路上的绊脚石。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#38; 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, 10000 MW</td>
			<td>ThermoFisher Scientific</td>
			<td>D-22911</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Alexa Fluor 568, 10000 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，它已广泛在文献中用于与认知过程干扰，而无需使用非常高的剂量，可能得到主要次级效应。例如，转录的阻断剂已被使用这种技术施用，以确定需要的基因的存储器相表达。这种分子的胸部注射与生存兼容数天27，这意味着，对其他目标的潜在毒性作用，可以限制，只要是公选择的浓度。在这样的条件下，可以得到在存储器的选择性和时间依赖性效应，从而显示出脑（ 图2）的有效定向。 分子进入头血淋巴的扩散导致快速，剂量依赖性的效果。 单眼注射是一种方法，以使感兴趣的分子的快速扩散到通过血淋巴整头，特别是如果他们可以具有在大脑许多普遍的目标。用这种方法来管理allatostatins，神经肽也可作为神经激素28）。其结果是，在嗅觉学习试验中观察到降低的性能，具有一致的在参与嗅觉处理不同脑区allatostatin受体和学习28的建议的存在。这种效应的剂量依赖性曲线可以通过注入不同浓度的并行运行（ 图3），独立组来建立。 管理方式不同，产生对脑功能类似的效果。 依米丁，蛋白合成的阻断剂，用于削弱的早期嗅觉记忆长期记忆，通常是调节后1-2天数表示的形成。在大多数已发表的研究已经注入到胸部29。我们发现，类似的效果可以通过经由单眼道直接施用它到大脑得到（ 图4）:提供的注射参数（更小的体积的调整，高浓度和更短的调理前延迟），我们获得的降低（约20％），类似于在使用相同的药物量（10 nm）的文献中发现。 -在Stollhoff 等人 ，2005 29与图4进行比较。 局部注射的效果被限制在时间和空间为了测试注射到特定的大脑区域毒品的时空特性，利用，蜜蜂嗅觉PER调节模式进行了培训，然后在蘑菇体肾盏或垂直波瓣0.5 NL 740毫米普鲁卡因（一种麻醉剂）的双侧注射（盐水用作对照）。当蜜蜂依次测试召回1,2和注射后3小时，性能与双边注射入裂片（ 图5）蜜蜂仅受损。从波瓣完好的神经输出，而不是从肾盏，已知是必要的嗅觉记忆检索，所以这表明普鲁卡因仍然本地化到其中已经注射了至少3小时的叶。这也表明，当注射到肾盏，扩散到附近的波瓣被同期限于以上，由于普鲁卡因的calycal注射没有导致凸起的封锁。 以下药品监督管理行为的表型往往是上下文相关 以前的实验已经表明，与可卡因蜜蜂治疗后高估蔗糖溶液10,30的质量。要查看是否这种效果是依赖于上下文（在这里，基线蔗糖质量），自由飞行的蜜蜂用挥发可卡因治疗。分别标记自由飞行的蜜蜂被允许在含1M蔗糖溶液饲养觅食。在除去饲养，蜜蜂轻轻抓获50毫升离心管，因为他们要下车从进纸器。蜜蜂用游离碱的可卡因或车辆控制（蒸发乙醇）​​的任一100微克治疗。处理后，将蔗糖馈线要么通过一个0.5 M或2.0M的蔗糖馈线替换，并且率觅食返回到馈线被记录下来。使用这种模式，可卡因处理蜜蜂在0.5M的馈线增加了其觅食的努力，但不是在2.0M的进纸器（ 图6）。在与两个蔗糖浓度看到效果的差很好地说明了采取环境线索考虑学习蜂行为时的重要性。 <img alt="图1" src="/files/ftp_upload/54695/54695fig1.jpg" /> 图 1:注射部位的激光共聚焦扫描图像 。的Alexa 546标记的葡聚糖与药物溶液（红色）一起注入。要识别神经毡SA反染色DAPI添加（绿色）。在右半球中的注射位点位于在垂直波瓣（VL），显示为一个成功的注射的例子。在左半球注射部位位于环神经纤维的垂直波瓣，示为一个不成功的喷射的实例的背侧。比例尺= 100微米，MB:蘑菇体，AL:触角叶，D:背，V:腹侧，L:左，R:右键<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54695/54695fig2.jpg" /> 下面食欲嗅觉康迪放线菌素D（转录阻滞剂）对长期记忆，当注射到胸部时间相关效应在不同的延迟: 图 2。<html> tioning（6,9或12小时），1微升放线菌素D（1.5毫溶于PBS中）注射到胸腔。长期记忆（LTM）检索，评估，3天调理后（N = 25 - 65）。存储器性能以时间依赖的方式减少，相比于PBS处理控制:效果显著当注射过放置6小时空调（χ2 = 18.04，P &lt;0.005）后，而不是在较长的延迟（ 9 H:χ2 = 0.95; 12小时:χ2 = 0.47），这表明LTM形成需要一个波转录该调理后在规定的时间窗口期间发生。误差棒代表标准误差。数据此前公布的27，经许可在这里重现。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4695 / 54695fig3.jpg"/&gt; 图 3:学习绩效的剂量依赖性抑制继神经肽的单眼注射 allatostatin C中的神经肽注入头血淋巴（200 NL在PBS），通过中值单眼，嗅觉调节前1小时。用不同浓度的注入（或PBS控件）动物的独立团体进行了培训。 Allatostatin C处理导致了学习性能降低，如通过的条件的反应在最后调理百分比以下一U形曲线（N = 70-78）评估，以剂量依赖的方式。这一下降是在其他浓度在10 -6 M显著但不是。误差棒代表标准误差。数据此前公布的28日 ，经许可在这里适应了。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig3large.jpg" target="_blank">请点击此处查看大版本</a>这个数字的锡永。 <img alt="图1" src="/files/ftp_upload/54695/54695fig4.jpg" /> 图 4:1（D）AY存储器 的阻断 之后通过单眼道依米丁（翻译抑制剂）的注射液的蛋白质合成抑制剂依米丁（50毫在PBS，200 NL）注射入脑，通过单眼道，20分钟前嗅觉调节。那么内存进行测试24小时后。治疗显著损害记忆力（χ2 = 7.03，P &lt;0.01）相比，PBS处理的对照组（N = 57-70）作为。误差棒代表标准误差。 JM Devaud，未发表的数据， <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig4large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图1"的src ="/文件/ ftp_upload / 54695 / 54695fig5.jpg"/&gt; 图 5:解剖和显微注射的时空特异性继食欲嗅觉空调，普鲁卡因双侧注入无论是蘑菇体肾盏或垂直波瓣。存储器检索被评估注射后1小时，并仅受普鲁卡因注射到叶（处理后1小时:相对于盐水:χ2 = 10.00，P &lt;0.005;相对于普鲁卡因到盏:χ2 = 32.92，P &lt; 0.005）。的影响可能仍然可以看到注射后2小时（χ2 = 6.65，P &lt;0.01），3（χ2 = 27.22，P &lt;0.005），并且仍然是位置特殊（2小时:χ2 = 8.60，P &lt; 0.05; 3小时:χ2 = 17.15，p &lt;0.0001），表明仅注入面积受普鲁卡因。比例是相对于调理级杜响最后审判调理。误差棒代表标准误差（N = 23-28）。数据此前公布的31日 ，经许可在这里重现。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54695/54695fig6.jpg" /> 图 6:对自由飞行蜜蜂可卡因的影响探视率（由试验期间的所有蜜蜂蜜蜂定/平均参观访问次数）已增加在低质量源挥发可卡因治疗（0.5M:-T 70 = 5.0710，p = 0.00003），但不能以高品质的源（2M:-T 70 = -0.2087，p值= 0.8353）。方框表示1 次和第 3 次与显示位数中线四分位数。胡须扩大到1.5X中的四分位范围。因为所有的各个数据点被叠加离群未绘出。数据此前公布的10，经许可在这里重现。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54695/54695fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 治疗 </td><td> 可以用自由飞行蜜蜂做些什么呢？ </td><td> 优点 </td><td> 缺点 </td></tr><tr><td>口服药物治疗</td><td>是。 </td><td>很简单，微创。 </td><td>蜜蜂消化并不简单</td></tr><tr><td>局部治疗</td><td>是。 </td><td>简单，微创，快捷。 </td><td>重复治疗可能会有问题。 </td></tr><tr><td>注射到胸腔</td><td>复杂，影响蜜蜂飞行能力</td><td>一致的和健壮。 </td><td>有点侵入。潜在的伤害/压力蜜蜂。 </td></tr><tr><td>注入位数单眼</td><td>不建议。 </td><td>一致的和健壮，有点本地化。 </td><td>有点侵入。潜在的伤害/压力蜜蜂。 </td></tr><tr><td>注入单眼道</td><td>不建议。 </td><td>非常本地化</td><td>非常侵入。潜在的伤害/压力蜜蜂。 </td></tr><tr><td>微注射到大脑区域</td><td>不建议。 </td><td>非常本地化</td><td>非常侵入性的，难以执行。潜在的伤害/压力蜜蜂。 </td></tr><tr><td>挥发的药物传递</td><td>是。 </td><td>简单，微创，快捷。 </td><td>所有药物不起作用。 </td></tr></tbody></table> 表 1:Compari治疗方法不同及性能的儿子。

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

9.8K 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

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...