我们感谢奥斯曼Kaftanoglu的拍摄过程中有益的建议和帮助。我们要感谢匿名审稿人有见地的意见。这项工作是由挪威研究理事会（补助180504，191699，213976和），玛丽Curie/FP7（项目编号。238665），国家老化研究所（NIA授予P01 AG22500）和皮尤慈善信托基金的支持。

1。从实际年龄衰老脱钩本节介绍的双队列的殖民地，它包括确定个人共享同一个实足年龄（"单年龄组"）和巢蜜蜂的队列的队列的设置。单年龄组的同年龄人最终会分成不同的工人类型不同老化动力学 - 这些都是护士与蜜蜂放缓，采集蜜蜂与加速功能下降。所有的程序都描述一个实验殖民地。然而，我们建议，进行实验用于至少两个菌落复制，使细胞集落的影响可以被控制为（2 - 复制 - 设计）。 <ol><li> 准备蜂巢箱为双队列殖民地:准备一台普通的蜂巢框，接收两个食品梳子蜂蜜，花粉另一种食物梳子，和两个空的梳子。一定要找到一个交配女王，以及一个供体的殖民地超过3,000窝蜜蜂。双方将在后面介绍（1.3）。 </li><li> 获取和标记相似的实足年龄人士:收集梳子与密封育雏，即将涌现。对于一个复制预期收回梳子总3,000-5,000上限育雏细胞。对于每个复制使用育雏的平衡量从至少三个不同的蜂巢来源，以避免母体基因型（蜂巢原点）的偏斜分布。 <ol><li>将育雏梳子在孵化器设定为34℃，60％-70％的相对湿度。确保存储梳子以这样一种方式，新兴育雏无法逃脱。 </li><li>让蜜蜂出现两天并与胸廓（ 如统一POSCA，三菱铅笔有限公司）小漆标签标记这些蜜蜂。油漆标记将允许识别单个年龄组（出苗天）的蜜蜂，并从其他重复的菌落区别开来。 </li></ol></li><li> 设置一个双队列殖民地，包括i的队列dentified，单身年龄蜜蜂:在一天年轻的蜜蜂已经被标记，收集从捐赠者的殖民地约2500-3000巢蜜蜂（比较第1节中讨论。），并添加这些标记的蜜蜂来之前准备了蜂巢箱（见步骤1.1）。后者个人将构成不明巢蜂队列。 <ol><li>添加王后，这将是最初仅限于女王笼（市售）。密封笼食用糖果（ 如 Apifonda，Südzucker公司，曼海姆/ Ochsenfurt，德国），使工蜂慢慢松开女王。 </li><li>添加新出现的和显着的蜜蜂，这将构成一个年龄组。这些蜜蜂是唯一显着的个体，并为以下所有步骤的焦点小组。 </li></ol></li><li> 监测发病觅食和标记征粮:为了评估发病和护士对觅食的单年龄组的过渡动态，监测觅食活动辰人口统计发展很一天每个殖民地。开始计数5天后菌落进行设置（ 图1）。 <ol><li>计数蜜蜂从内按固定时间3×20分钟观察期间觅食航班（入口计数）返回的总数。请务必在定向航班期间不计蜜蜂（见讨论）。 </li><li>当入口计数表明相当觅食活动开始（&gt; 100入口数/天），开始标记觅食。要做到这一点，单年龄组（单标记个体）的觅食后，收到来自他们的第一觅食航班返回第二个油漆标记。这个标志漆将指定觅食发病的当天，并且将允许以后识别觅食年龄每觅食。 </li><li>每日重复标记直到蜜蜂足够数量的已标记。用于估计标记觅食足够数量的，期望的不超过5％-10％的检索率这些蜜蜂已经老化后，通常经过14天觅食。 </li></ol></li><li> 取样:由于所有的初始标记蜜蜂具有类似的实足年龄，护士和老觅食的年龄匹配的基团，可以同时采集，当觅食觅食已为≥14天。 <ol><li>单标记的护士蜜蜂蜂房内收集，并通过护理行为（喂食和清洗幼虫在开放育雏细胞放下头）确定。 </li><li>双标征粮也收集蜂巢内每天觅食活动开始之前。 </li><li>收集蜜蜂提供充足的通风笼（管，盒），并保持黑暗，直到进一步的处理。另外，对于转录组，表观遗传或蛋白质组学研究，直接扣在液氮中冷冻蜜蜂。收集个人的均衡数量从所有测试组和复制的殖民地。 </li></ol></li></ol> 2。与快速与缓慢老化工人工人通过改变蜂房的人口&lt;逆转/ P&gt; 本节详细介绍如何逆转，从工人的加速老化（觅食）工人与减缓老化（护士蜜蜂）执行。这种行为逆转诱导，觅食时遇到一个缺乏护士的蜜蜂，通常从事育雏舍。在回归过程将分离的单个菌落复制成两个蜂箱:一个配置单元的护士蜂分数（"护士衍生"），另一个与觅食分数（"觅食来源"）。后成功逆转，塑料可能出现的症状和逆转衰老可以研究在单一年龄组与恢复工人，继续征粮，持续护士蜜蜂和新招募的觅食。和以前一样，确定了单年龄组，而不是不明巢蜜蜂队列的蜜蜂，构成了实验焦点小组。 <ol><li> 制备方法:复制荨麻疹护士（单标）和征粮（双标）是在上一节中所述提供。确保不复归开始，每个重复菌落小于500标觅食，以确保有足够的检索复归完成后。 <ol><li>逆转后的安全鉴定测试组的关键是整个觅食人口在原来的蜂巢已经回归之前被标记为关闭。下面的步骤描述为一个复制。 </li><li>回归前一天，准备一个额外的蜂巢箱为觅食衍生蜂房（见步骤1.1）。找到两个皇后和两个育雏梳子捐助荨麻疹。转移到实验前殖民地刷了所有的成年蜜蜂从这些梳子。一笼女王（见步骤1.3），以及一个育雏梳将在原有的蜂巢箱代替女王和育雏梳子。另一组的女王和育雏梳将要使用的第二天新蜂巢框。新的育雏梳子和外来皇后两个，原来和新蜂巢建议，以确保类似的分配分开觅食和护士s将会同样经历改变了蜂房线索（"蜂巢闻"）。 </li></ol></li><li> 逆转:早晨，就在回归前，笼女王和育雏梳添加到新的方块，将接收觅食衍生部分。等到觅食高峰时开始。然后将原来的殖民地注明来源为觅食和护士的蜜蜂至少有100米的距离原来的位置。 <ol><li>在原来的位置上，建立了觅食来源的蜂房与育雏，只有女王的新盒子。 </li><li>征粮将离开原脱臼蜂巢框，头回原来的位置。允许觅食返回到2小时的原始位置，以达到下一个完成觅食人口从巢蜂分离。 </li><li>然后，终止分离，关闭掉原来的，现在的"护士衍生的"配置单元，它至少有3公里远转移到一个养蜂场。 </li></ol></li><li> 蜂巢维护和监控对于成功的社会任务反转:定期检查实验荨麻疹健康，开放的育雏。 <ol><li>在殖民地操纵后的第一天更换无人值守和死育雏，以减少潜在的病原体负荷。 </li><li>为了验证成功逆转的觅食来源的蜂房内，采取育雏梳子的图片介绍这些之前，再次当梳子被替换或者当复归试验完成（ 图2）。与以前不封顶的育雏和开放，现场育雏区域是护理活动中觅食的集落可靠标记。 </li></ol></li><li> 采样:觅食和护士后3-8天可以检测伴随社会反转生理效应分离。 <ol><li>我们建议所有取样试验组， 即恢复工人和持续觅食（觅食衍生蜂巢），以及持续的护士和新招聘的觅食（护士衍生蜂巢）8日内开始逆转后。 </li><li>关口如步骤1.5中所述课样品。 </li></ol></li></ol> 3。由脂褐质的定量分析工型特异性细胞衰老模式脂褐质是细胞衰老的一个普遍的生物标志物。作为一个内在的累积产物，脂褐质的特定的自体荧光（发射最大 = 530-650纳米），可用于检测18。 <ol><li> 解剖和固定:冰上冷藏蜜蜂，直到一动不动，删除并剖析出所需的组织样本。 <ol><li>在4℃下过夜孵育;转移到固定剂（pH 7.2的4％多聚甲醛的磷酸盐缓冲盐水，PBS）中</li><li>在PBS中洗涤样品3次。 </li></ol></li><li> 组织处理和安装:通过使用振动切片机刀片， 例如莱卡VT 1000S（徕卡生物系统公司，旅游Nussloch，德国）切割的组织样品与不超过40微米厚的切片，例如。 <ol><li&gt;对于结算，过夜孵育组织切片在30％甘油中（PBS），和另外2小时，在50％甘油中（PBS）中。 </li><li>安装在显微镜玻片切片在50％甘油（PBS）中。对于长期储存密封盖玻片指甲油。 </li></ol></li><li> 图像采集:为了检测脂褐素，我们建议使用激光扫描共聚焦显微镜，提供激光线，λ= 514，561nm或类似的激励，并与探测器的带宽设置为570-650纳米。 <ol><li>为了更好地识别脂褐素，包括第二通道，并做了同步扫描在更短的波长光谱（激发= 405 nm处;排放量= 410-450纳米）。较长波长信道将揭示两者，脂褐素颗粒，但也非特异性的"背景"，由于自身荧光气管等非粒状结构。第二，较短的波长信道将只显示非特异性自体荧光，但不脂褐素。因此，脂褐素标识可以是facili<html>通过比较两个通道的信号，只有其中一人流露颗粒与脂褐素特异性荧光tated。 </li><li>对采集的高分辨率图像使用目的与40X放大率或更高，以及1.25或更高优选的数值孔径。扫描图像叠加约100×100×10μm的3个维度。每一个个人和组织样本必须由多个图像堆栈来表示。 </li><li>为了减少因技术变化个体内和个体间变异，始终保持激光功率和探测器灵敏度恒定。 </li><li>由一天到一天的技术变化减少偏见扫描每个数扫描会话的所有测试组的相等样本数。 </li></ol></li><li>图像处理:使用软件包的模块，允许对显微图像堆栈先进的加工，例如ImageJ的（健康，马里兰州贝塞斯达，美国美国国家研究院​​，gej.nih.gov / IJ /"目标="_blank"&gt; http://imagej.nih.gov/ij/）。 <ol><li>生成一个二维最大投影为每个3D图像堆栈。 </li><li>为了衰减高频噪声，并保持结构与脂褐素颗粒的尺寸适用于一个高斯滤波器具有适中的籽粒大小。 </li><li>合并两个颜色通道，以方便识别脂褐素（见步骤3.2）。 </li></ol></li><li> 图片分析:确保执行量化步骤的主题将是盲目的测试群体认同。 <ol><li>对于所有的图像，先选择感兴趣区（ROI），涵盖了相关结构，并具有类似的尺寸从其他图像中感兴趣区域的区域。 </li><li>然后选择代表每个ROI脂褐素颗粒的期望数量。当选择一个ROI内脂褐素颗粒，下列规则的应用将减少主观偏见。 <ol><li>选择一致的位置，用于选择所述第一麻粒乐。这可以是，例如，一个ROI的最左边，并且该颗粒是最接近的边缘将始终是第一选择。 </li><li>此起彼伏，颗粒选择是最接近先前的选择（下一个邻居）。 </li><li>当选择一个邻居，只移动到一个方向，例如只能从先前的选择搜索的权利。这条规则可以防止该评选是由密密麻麻的颗粒偶尔集群为主。 </li><li>当选择完成后，通过概述和测量各颗粒面积评估每个脂褐素颗粒的大小。使用适当的统计测试，以比较不同的测试组的个体。 </li></ol></li></ol></li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+learning+deficits+can+be+reversible+in+honeybees+Apis+mellifera.">Age-related learning deficits can be reversible in honeybees Apis mellifera.</a> Exp Gerontol. 47, 764-772 (2012).</li><li>Capaldi, E. A., 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=Ontogeny+of+orientation+flight+in+the+honeybee+revealed+by+harmonic+radar.">Ontogeny of orientation flight in the honeybee revealed by harmonic radar.</a> Nature. 403, 537-540 (2000).</li><li>Marco Antonio, D. S., Guidugli-Lazzarini, K. R., do Nascimento, A. M., Simoes, Z. L., Hartfelder, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+silencing+of+vitellogenin+gene+function+turns+honeybee+(Apis+mellifera)+workers+into+extremely+precocious+foragers.">RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers.</a> Naturwissenschaften. 95, 953-961 (2008).</li><li>Whitfield, C. W., Cziko, A. -. M., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+in+the+brain+predict+behavior+in+individual+honey+bees.">Gene expression profiles in the brain predict behavior in individual honey bees.</a> Science. 302, 296-299 (2003).</li><li>Schmidt, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attraction+of+reproductive+honey+bee+swarms+to+artificial+nests+by+Nasonov+pheromone.">Attraction of reproductive honey bee swarms to artificial nests by Nasonov pheromone.</a> Journal of Chemical Ecology. 20, 1053-1056 (1994).</li><li>De Moraes, R., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+cell+death+in+the+hypopharyngeal+gland+of+the+honey+bee+(Apis+mellifera+L).">Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L).</a> Cell Biol Internat. 24, 737-743 (2000).</li><li>Sheehy, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow-cytometric+method+for+quantification+of+neurolipofuscin+and+comparison+with+existing+histological+and+biochemical+approaches.">A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches.</a> Arch Gerontol Geriatr. 34, 233-248 (2002).</li><li>Hsieh, Y. S., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+trophocytes+and+fat+cells+as+target+cells+for+cellular+senescence+studies.">Honeybee trophocytes and fat cells as target cells for cellular senescence studies.</a> Exp Gerontol. 46, 233-240 (2011).</li><li>Wolschin, F., Munch, D., 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=Structural+and+proteomic+analyses+reveal+regional+brain+differences+during+honeybee+aging.">Structural and proteomic analyses reveal regional brain differences during honeybee aging.</a> J Exp Biol. 212, 4027-4032 (2009).</li></ol>

我们什么都没有透露。

在这里，我们采用前面所述的方法8,16,17,19,20，并将它们集成到一个单一的工作流程，这将有利于灵活学习老龄化蜜蜂。我们的目标是为科学家是新手，这个领域与标准化的工具集，以取得相关样本材料，并改善不同的研究小组之间的实验重现性。虽然我们的程序简化，不需要特殊设备，在前面的描述（比较例如8），预防的一些措施，建议及以下收集。 从...

高度社会化的动物的复杂的菌落结构通过生殖种性的相互作用维持，并且通常是非再现工人的辅助阶级不同社会任务的行为。在不同的工人，具体的生理适应性使不同的同胞关心的行为，也与极端的寿命差异。蜜蜂和鼹鼠代表了最发达的动物模型，研究如何社会性链接到的加速，可以忽略不计或逆转老化的1-3模式。 在蜜蜂的殖民地，一个产蛋女王是由成千上万的工人，往往育雏，饲料的粮食援助，并参与防守，体温调节或卫生行为4。在这些工人中是极其短暂的征粮，护士蜜蜂与中间，和冬季（diutinus）与蜜蜂的寿命最长。个人，但是，不会永久绑定到一个特定的窝rker型，但会显示一条灵活的行为个体发育:他们从一个社会任务的行为更改为另一种（"时间种姓"）。乳臭未干的蜜蜂可以换到育雏抚育护士蜜蜂，最终可能会改变外界觅食。然而，乳臭未干巢蜜蜂也可以变身为最长寿的冬季蜜蜂和短命觅食，甚至可以恢复到通常寿命更长护士。极端（冬季蜜蜂）和中级（保姆蜂）寿命工人有发达的食品生产和贮藏器官与丰富的资源-而不是短暂的觅食（1,5审查）。然而，个人寿命的调节超越于个人的资源平衡简单的修改建议通过在卵黄蛋白，它具有不同的适应功能，在非再现工人等级，比如果冻生产6，激素控制7，免疫研究8，抗氧化剂防御9。 的f模式unctional下降（衰老）工人镜的寿命差距，为建立嗅觉，同时还为其他脑或运动功能10-13。具体地，仅在两个星期的觅食之后的显著下降学习函数相匹配类似的死亡率级数中觅食14，相对于缺乏可检测的下降（可忽略衰老）的长寿命的冬季蜜蜂15。 为了确定我们适应建立实验范式，使监测和处理老化型转变8,16,17灵活衰老的分子指纹。实验1的详细信息如何获取样品中的实足年龄和老龄工作者类型的具体的社会行为的影响可以分开。实验2描述了征粮与加速进入护士蜜蜂与减缓老化动力学的逆转。实验3提供了解剖学探测细胞衰老的影响的方法已建立的生物标志物细胞衰老（脂褐素）18升量化。

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Apifonda</td>
 <td>S&uuml;dzucker AG, Mannheim/Ochsenfurt, Germany</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>158127</td>
 <td></td>
 </tr>
 <tr>
 <td>phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>P4417</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Merck</td>
 <td>1.04094.1000</td>
 <td></td>
 </tr>
 </tbody>
</table>

obtaining specimens with slowed, accelerated and reversed aging in the honey bee model

高度社会化的动物的社会功能密切相关的个体之间的广袤的寿命差异。其中社会性昆虫，蜜蜂是最好建立的模型，以研究如何可塑性寿命和衰老是由社会因素来解释。 蜜蜂的工人等级包括护士蜜蜂，这往往育雏，并采集蜜蜂，负责收集花蜜和花粉。以前的工作表明，大脑功能和飞行性能的征粮衰老的速度比护士。然而，大脑的功能可以恢复，觅食时恢复到哺乳的任务。加快并逆转衰老的功能，例如模式都与代谢改变资源水平，在蛋白丰度的改变和免疫功能。卵黄，卵黄蛋白与激素控制和细胞防御适应功能，可作为中，控制工人的不同老化动力学网络的主要调控元件。在这里，我们描述了如何护士和觅食的出现可以被监控，和操作，包括逆转，从典型的短命觅食成更长寿命的护士。我们的代表结果表明，与同类实足年龄个体如何分化成蜂和护士蜜蜂实验条件下。我们举例说明如何行为逆转，从觅食回到护士可以进行验证。最后，我们显示如何不同细胞衰老可通过测定脂褐质的积聚，衰老的普遍的生物标记物进行评估。 为了研究可能连结社会影响的机制和老化的可塑性，该协议提供了一个标准化的工具集，以获取有关样品材料，并提高未来的研究中数据的可比性。

协议第1和2详细测试组如何能够获得学习的加速属性，减缓和扭转与一个单一的年龄组菌落老化。为了监视工人型分化，伴随着个体发育正常，我们评估觅食计数（"高考计数"）的6个菌落（ 图1，比较第1节）。该图显示，通常观察不到从护士相当大的变化的觅食状态前的人更超过10天。在觅食计数标记变异观察与问候觅食不同群体之间发生的时机，作为一个显着的一天到一天的每一个?...

Watch this Scientific Journal Video about Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model at JoVE.com

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

在蜜蜂的工人，老化依赖于社会行为，而不是实际年龄。在这里，我们将展示如何工人种类具有非常不同的老化模式可以得到和细胞衰老分析。

获取标本减慢，加速和反老化的蜜蜂型号

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

obtaining-specimens-with-slowed-accelerated-reversed-aging-honey-bee

Research

JoVE Journal

Biology

11.2K 次观看.  Norwegian University of Life Sciences. 在蜜蜂的工人，老化依赖于社会行为，而不是实际年龄。在这里，我们将展示如何工人种类具有非常不同的老化模式可以得到和细胞衰老分析。 

视频: Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

脑银行业：使你的研究样本中，大部分

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

科研

生物学

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 ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

解剖和蜜蜂背血管的观察心功能的研究

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

蜜蜂致死农药暴露对蜂群水平的影响

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

用于大黄蜂（Bombus）蜂巢入口的3D打印花粉陷阱

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

小尺寸远程模型日本美达卡（奥里齐亚斯拉蒂普斯）的果纳切除术和血液采样程序

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

获取标本减慢，加速和反老化的蜜蜂型号

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