这项工作是支持由德国联邦教育和研究部（BMBF）在伯恩斯坦焦点授予：学习（BFNL）DE神经元的基础，我们感谢门泽尔的手稿的早期版本上的宝贵意见Randolf。

1。捉蜜蜂从蜂巢<ol><li>实验开始前一天，2至下午4时，蜜蜂离开蜂房都一网打尽。要做到这一点，一个UV光透水有机玻璃金字塔（= 30厘米，先端3,5，5 × 3厘米，底座18 x 18厘米），这是在心尖区和基地的Closable，是在20日举行的高度 - 基地开放和心尖蜂巢的入口前的30厘米的距离，使蜜蜂离开蜂房进入金字塔底部封闭。然后关闭该基地，并带来拍摄的蜜蜂实验室作进一步的处理。 </li></ol> 2。从金字塔蜜蜂转移到玻璃小瓶<ol><li>在实验室中，金字塔是放在底座上的。金字塔的墙壁是黑暗的（如用毛巾），但发现是左心尖。由于其积极的趋光性，蜜蜂会离开金字塔的顶点时打开。一个接一个，蜜蜂转移到玻璃小瓶从金字塔开放顶点的小瓶。蜜蜂每一个小瓶。因此，Apex是封闭的，当蜜蜂进入小瓶。 </li></ol> 3。在管治理的蜜蜂<ol><li>蜜蜂被固定在2.5-3.5分钟冰的玻璃小瓶冷却。这是最好看蜜蜂，从冰中删除，只要它停止不动。 </li><li>一个单一的固定蜂是利用胶带在一个小塑料管，这样，它是能够自由移动它的长鼻，但不是它的头部，胸部或腿部。重要的是，颈部是没有压缩。 </li><li>更好的处理和识别的机架上，固定在一个塑料管每蜜蜂被放入一个编号钻孔。它已经为空调或内存检索删除后，管总是返回完全相同的钻孔。 </li></ol> 4。饲喂蜜蜂<ol><li>在第一天晚上（下午4:00-6:00），被抓后，蜜蜂是美联储满足蔗糖溶液（0,88男，白色的精致家庭糖溶解在自来水）。为了养活一只蜜蜂，其人均蔗糖溶液通过触摸其触角和动物是可以消耗一个液滴的蔗糖溶液（4μL）引起。蜜蜂已经厌倦了一个又一个，直到他们不再表现出快速和可靠的的每时，他们的触角与蔗糖溶液感动。 </li><li>在随后的每次实验蜜蜂晚上（下午4:00-6:00）美联储1蔗糖溶液的液滴（4μL，0,88 M，白色的精制糖在解决自来水）后与另外四次之一。重要的是蜜蜂的天线或在喂养过程中的长鼻和涂污管不污染的蔗糖溶液，蔗糖溶液。蜜蜂不宜过饱或附近的空调网站排除的培训与蔗糖的刺激上下文关联的可能性。 </li></ol> 5。保持利用蜜蜂隔夜<ol><li>蜜蜂是在室温下过夜存放在一个塑料碗。自来水充入碗（约0,5-1厘米高）。利用蜜蜂的机架放置在一碗使用酶标板作为一个平台的水线以上。最后，碗是用纸板覆盖。 </li></ol> 6。嗅觉空调嗅觉调节，在实验的第一天早上（上午10时）进行。条件刺激（CS）是一种气味，无条件刺激（美国）的蔗糖溶液。 <ol><li> 4μL的气味（如丁香油）到一个圆形滤纸（直径1,3厘米），然后将20毫升注射器插入吸管。引擎盖下的气味是吸管和过滤嘴是用来防止污染的吸管。 </li><li>利用蜜蜂的机架放置在靠近空调的网站30分钟的调理过程开始之前，但在一定距离，以在单一的蜜蜂是空调的排气管前的地方。 </li><li>空调由三个配对的气味（条件刺激，CS）和与审判间的间隔为10分钟（ITI）的蔗糖溶液（无条件刺激，美国，1,25中号）。通过一个音频播放器的实验者，确保声音信号的刺激发病，刺激抵消，刺激的持续时间，并把精确的计时。收购审判开始与一个10秒的动物安置在排气前。前不久结束的10秒注射器中含有的气味是摆在前面，在蜜蜂的触角有针对性的蜜蜂3厘米。随后，气味是5秒，20毫升的空气通过注射器推。第3秒后，两个天线的远端鞭毛感动与蔗糖溶液蘸牙签和动物舔时间为4秒moistend牙签。 13秒后，CS的刺激结束后，蜜蜂取出的培训方面，并放置在机架的背面。一个培训试验共持续28秒。 重要的是，无论是蜜蜂的触角也没有的长鼻蔗糖空调后覆盖。因此，必须确保，牙签只蘸蔗糖溶液，无滴，牙签上的蔗糖溶液的形式。 </li><li>调理后的衣架被放置在碗里。 </li><li>在实验动物的行为，即发生在放置过程中，CS和美国文稿的PER，监测，并指出实验者。 </li></ol> 7。内存保留<ol><li>保留测试可以在任何时间间隔（分钟到几天）。内存测试开始前，与蜜蜂的机架放置在靠近空调的网站30分钟。 </li><li>内存测试由5秒CS如果没有美国的演示介绍。测试开始用10秒的动物安置在前面的排气。随后，气味是5秒，如上面所述。动物的行为，即在安置过程中发生的PER和CS简报指出，在实验。 </li><li>在实验结束长鼻延伸响应再次引起与蔗糖溶液接触的触角，以确保动物仍然能够延长它的长鼻。 </li></ol> 8。全身注射液全身注射的时间点取决于实验设计。 <ol><li>在上述使用一次性注射针（21 G）的飞行肌肉17 scutal裂缝旁边的鳞甲后部cuticula有一个小孔。 </li><li>使用玻璃毛细管，1μL的解决方案是通过孔注入，在进入飞行肌肉cuticula。训练有素的实验者可以注入一个蜜蜂，每30秒，允许精确定时注射液和调理。 </li></ol> 9。在实验过程中喂养蜜蜂<ol><li>调理后的晚上（下午4:00-6:00），蜜蜂喂1蔗糖溶液的液滴（4μL，0,88 M，白色的精制糖溶解在自来水中）后与另外四次之一。重要的是蜜蜂的天线或在喂养过程中的长鼻和涂污管不污染的蔗糖溶液，蔗糖溶液。蜜蜂不宜过饱或附近的空调网站排除的培训与蔗糖的刺激上下文关联的可能性。 </li></ol> 10。数据收集和数据分析<ol><li>在实验过程中出现的长鼻扩展响应监测。蜜蜂是取得了积极的，如果延伸至CS的发病和美国的演示（培训期间），或在CS演示（在测试阶段）的长鼻，穿越打开下颌骨之间的虚拟线。 </li><li>要在分析动物的，必须符合两个条件：他们必须生存在整个实​​验，他们必须证明在实验结束后无条件长鼻延伸反应（PER）的蔗糖溶液。蜜蜂采集和保留测试期间表现出的PER人员的比例是绘制每个CS演示。 </li></ol> 11。代表性的成果： 这里给出了两个实验。 在第一个实验中，我们期待在全身注射磷酸盐缓冲液（PBS的影响;在MM：137氯化钠，2,7氯化钾，10,1娜 2 HPO 4，1,8 KH 2 PO 4，pH值7， 2）对学习和长期记忆的形成。三组的蜜蜂CS - 中美三个配对试验间间隔10分钟的训练：非治疗组，1μL，PBS在训练前30分钟注射组，和假注射组治疗同样的方式，PBS注射组，但没有注射PBS。训练内存后24小时进行了测试，一个CS在所有三组的演示。在空调条件反应的动物的比例显着增加在三个培训试验（图1，为的因素，时间的反复测量方差分析F 2378 = 340456，P &lt; 0,05）。每无差异，可以观察到三组之间在空调（ANOVA反复测量的因素群体F2，189 = 1299，P&gt; 0.05）。这拥有一个24小时保留测试（F2，187 = 0752，P&gt; 0.05）真正的比较时，用PBS注入和假注入动物的非治疗性的动物。 在第二个实验中，我们表现出抑制记忆保持三和四天训练后，当蛋白质合成抑制剂茴香霉素（10毫米）系统注入30分钟后，CS - 中美三个配对。茴香霉素注射组与PBS注射组比较，揭示了在保留吨之间显着性差异EST（图2，方差分析重复测量因子F1组，94 = 8,86，P &lt;0,05）。可以观察到在3天4天的内存测试的一个主要区别。 Wüstenberg 等类似的结果。18，谁也空调蜜蜂CS -中美三个配对，尽管不同的协议。 <img src="/files/ftp_upload/2282/2282fig1.jpg" alt="图1" /> 图1。 Cuticula病变或PBS注射不会改变收购，并记忆保持。动物CS中美三个配对（收购）的培训。 24 h后，内存测试（内存保留）。一个洞前三十分钟的训练，是在两个组cuticula（病灶，PBS），这些群体之一是用1μLPBS（PBS）的注入。第三组是原封不动的（不治疗）。主办的动物是每个组每气味演示的百分比。方差分析没有透露在收购阶段群体之间的任何显着性差异（F2，189 = 1.299，P&gt; 0.05），或保留在内存（F2，187 = 0.752，P&gt; 0.05）。 <img src="/files/ftp_upload/2282/2282fig2.jpg" alt="图2" /> 图2。注射茴香霉素40分钟后，最后的CS美配对减少内存保留3天，4天训练后，训练与CS中美三个配对（收购）和1μL，10 mM的茴香霉素（各向异性过滤）或1μLPBS注入动物1小时后的第一次训练试验。内存测试（内存保留）24小时，48小时，72小时和96小时后。主办每组每气味介绍动物的百分比。茴香霉素注射导致的记忆保持72小时和96小时的训练后显着减少（F1，94 = 8.86，P &lt;0.05），方差分析显示。

<ol>
	<li>Menzel, R., Giurfa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+architecture+of+a+mini-brain:+the+honeybee.">Cognitive architecture of a mini-brain: the honeybee.</a> Trends Cogn Sci. 5, 62-71 (2001).</li><li>Menzel, R., Leboulle, G., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+brains,+bright+minds.">Small brains, bright minds.</a> Cell. 124, 237-239 (2006).</li><li>Menzel, R., Kesner, R. P., Olton, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurobiology of comparative cognition. , (1990).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical-Conditioning+of+Proboscis+Extension+in+Honeybees+(Apis-Mellifera).">Classical-Conditioning of Proboscis Extension in Honeybees (Apis-Mellifera).</a> J. Comp. Psyc. 97, 107-119 (1983).</li><li>Menzel, R., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+in+honeybees:+from+behavior+to+neural+substrates.">Learning and memory in honeybees: from behavior to neural substrates.</a> Annu. Rev. Neurosci. 19, 379-404 (1996).</li><li>Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+dynamics+in+the+honeybee.">Memory dynamics in the honeybee.</a> Journal of Comparative Physiology A-Neuroethology Sensory Neural and Behavioral Physiology. 185, 323-340 (1999).</li><li>Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+the+memory+trace+in+a+mini-brain,+the+honeybee.">Searching for the memory trace in a mini-brain, the honeybee.</a> Learn. Mem. 8, 53-62 (2001).</li><li>Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+formation+in+the+honeybee+(Apis+mellifera)+and+its+dependency+on+the+cAMP-protein+kinase+A+pathway.">Learning and memory formation in the honeybee (Apis mellifera) and its dependency on the cAMP-protein kinase A pathway.</a> Animal Biology. 56, 259-278 (2006).</li><li>Hourcade, B., Muenz, T. S., Sandoz, J. C., Rossler, W., Devaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+memory+leads+to+synaptic+reorganization+in+the+mushroom+bodies:+a+memory+trace+in+the+insect+brain.">Long-term memory leads to synaptic reorganization in the mushroom bodies: a memory trace in the insect brain.</a> J. Neurosci. 30, 6461-6465 (2010).</li><li>Denker, M., Finke, R., Schaupp, F., Grun, S., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+odor+learning+in+the+honeybee+antennal+lobe.">Neural correlates of odor learning in the honeybee antennal lobe.</a> Eur. J. Neurosci. 31, 119-133 (2010).</li><li>Szyszka, P., Galkin, A., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+and+non-associative+plasticity+in+kenyon+cells+of+the+honeybee+mushroom+body.">Associative and non-associative plasticity in kenyon cells of the honeybee mushroom body.</a> Front Syst. Neurosci. 2, 3-3 (2008).</li><li>Okada, R., Rybak, J., Manz, G., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning-related+plasticity+in+PE1+and+other+mushroom+body-extrinsic+neurons+in+the+honeybee+brain.">Learning-related plasticity in PE1 and other mushroom body-extrinsic neurons in the honeybee brain.</a> J. Neurosci. 27, 11736-11747 (2007).</li><li>Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+activation+of+cAMP-dependent+protein+kinase+during+conditioning+induces+long-term+memory+in+honeybees.">Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees.</a> Neuron. 27, 159-168 (2000).</li><li>Locatelli, F., Bundrock, G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+and+temporal+release+of+glutamate+in+the+mushroom+bodies+improves+olfactory+memory+in+Apis+mellifera.">Focal and temporal release of glutamate in the mushroom bodies improves olfactory memory in Apis mellifera.</a> J. Neurosci. 25, 11614-11618 (2005).</li><li>Stollhoff, N., Menzel, R., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+recovery+from+extinction+depends+on+the+reconsolidation+of+the+acquisition+memory+in+an+appetitive+learning+paradigm+in+the+honeybee+(Apis+mellifera.">Spontaneous recovery from extinction depends on the reconsolidation of the acquisition memory in an appetitive learning paradigm in the honeybee (Apis mellifera.</a> J. Neurosci. 25, 4485-4492 (2005).</li><li>Stollhoff, N., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consolidation+of+an+extinction+memory+depends+on+the+unconditioned+stimulus+magnitude+previously+experienced+during+training.">Consolidation of an extinction memory depends on the unconditioned stimulus magnitude previously experienced during training.</a> J. Neurosci. 29, 9644-9650 (2009).</li><li>Snodgrass, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the honeybee. , (1956).</li><li>Wustenberg, D., Gerber, B., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+communication:+long-+but+not+medium-term+retention+of+olfactory+memories+in+honeybees+is+impaired+by+actinomycin+D+and+anisomycin.">Short communication: long- but not medium-term retention of olfactory memories in honeybees is impaired by actinomycin D and anisomycin.</a> Eur. J. Neurosci. 10, 2742-2745 (1998).</li><li>Vollbehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Orientierung+junger+Honigbienen+bei+ihrem+1.">Zur Orientierung junger Honigbienen bei ihrem 1.</a> Orientierungsflug. Zool. Jb. Physiol. 79, 33-69 (1975).</li><li>Scheiner, R., Erber, J., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+and+the+individual+evaluation+of+the+reward+in+honey+bees+(Apis+mellifera+L.).">Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).</a> J. Comp Physiol [A]. 185, 1-10 (1999).</li><li>Sanchez, B. r. i. t. o., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+studies+on+tarsal+gustation+in+honeybees:+sucrose+responsiveness+and+sucrose-mediated+olfactory+conditioning.">Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning.</a> J. Comp Physiol A Neuroethol. Sens. Neural Behav. Physiol. 194, 861-869 (2008).</li><li>Menzel, R., Manz, G., Menzel, R., Greggers, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massed+and+spaced+learning+in+honeybees:+the+role+of+CS,+US,+the+intertrial+interval,+and+the+test+interval.">Massed and spaced learning in honeybees: the role of CS, US, the intertrial interval, and the test interval.</a> Learn. Mem. , 198-208 (2001).</li><li>Friedrich, A., Thomas, U., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+at+different+satiation+levels+reveals+parallel+functions+for+the+cAMP-protein+kinase+A+cascade+in+formation+of+long-term+memory.">Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory.</a> J. Neurosci. 24, 4460-4468 (2004).</li></ol>

<ol><li>捕捉蜜蜂：要收集经验丰富的觅食的蜜蜂，蜜蜂被发现无论是在上午或下午，避免年轻蜜蜂方向的航班19时。觅食收集无论是花粉和花蜜。已经观察到的花粉和花蜜觅食之间的差异，根据他们的血糖反应能力和他们的学习能力20。为了赶上只有花蜜觅食，与蔗糖溶液填充一个人造的食物来源，可以安装附近的蜂巢和动物可以收集。回到蜂房花粉觅食，可以识别 20后腿的花粉筐。 </li><li>修复和处理：应避免冰蜜蜂延长冷却，因为它可能会影响他们的学习能力和生存，直到实验结束。跗管内仔细加以固定，以避免意外刺激蔗糖溶液。这是很重要的，因为蜜蜂已经证明，学习与succrose 解决方案3,21跗刺激。 </li><li>嗅觉空调：空调试验的数量可以是多种多样的，审间的间隔，测试时间点取决于重点研究3,22。 </li><li>喂养：蜜蜂的饱食水平是影响他们的学习表现和记忆保持 3,23 。这已规划的实验时，要考虑到。 </li><li>注射液：气囊是在位于蜜蜂胸部17。呼吸道系统的这些重要的部分是为生存所必需的。因此应注意不要损坏气囊注射过程中的。释放的气泡表明气囊损坏。 </li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Syringe Omnifix 20 ml</td>
<td>&nbsp;</td>
<td>B.Braun, Melsungen, Germany </td>
<td>4616200 V</td>
<td>&nbsp;</td>
<tr>
<td>White refined household sugar</td>
<td>&nbsp;</td>
<td>Kaisers Supermarket, Berlin, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Clove oil</td>
<td>&nbsp;</td>
<td>Bombastus, Freital, Germany </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Multipette plus</td>
<td>&nbsp;</td>
<td>Eppendorf, Hamburg, Germany</td>
<td>4981 000.019</td>
<td>&nbsp;</td>
<tr>
<td>Rack for 30 bee tubes</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic tubes</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>UV light-permeable plexiglass pyramid</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>height = 30cm, 
apex 3.5 x 3.5cm, 
base 18x18cm</td>
</tr>
<tr>
<td>Sticky tape</td>
<td>&nbsp;</td>
<td>Tesa, Hamburg, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillator sticks 1-5&mu;l</td>
<td>&nbsp;</td>
<td>Selzer, Driedorf-Roth, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillator tips 1-5&mu;l</td>
<td>&nbsp;</td>
<td>Selzer, Driedorf-Roth, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filter paper 0.16-0.2 mm thick</td>
<td>&nbsp;</td>
<td>Macherey-Nagel, D&uuml;ren, Germany</td>
<td>MN 616 or MN615</td>
<td>Adjusted to circular pieces with a 1.3 cm diameter</td>
</tr>
<tr>
<td>Disposable hypodermic needle</td>
<td>&nbsp;</td>
<td>B. Braun, Melsungen, Germany</td>
<td>4657683</td>
<td>0.45 mm diameter</td>
</tr>		</tbody>
		</table>
		</div>

behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (apis mellifera)

蜜蜂（ 蜜蜂 ）是众所周知的，他们的沟通和方向技能，并为他们令人印象深刻的的学习能力1,2。由于蜜蜂殖民地的生存取决于剥削的食物来源，采集蜜蜂学习和记忆变量花景点，以及他们的盈利能力。在自然环境中采集蜜蜂可以很容易地训练他们草料喂食地点和学习相关的信号，如气味或颜色的地方。食欲联想学习，也可以研究在实验室控制的条件下下，通过调理（PER），单独利用蜜蜂3,4长鼻扩展响应。这种学习范式使5-12在一个简单，高度可靠的的方式的学习和记忆的形成背后的神经细胞和分子机制的研究。行为药理学的方法是用来研究的分子机制。药物注入系统与特定分子的功能干扰，期间或之后的学习和记忆的形成13-16。 在这里，我们展示了如何培养每空调利用蜜蜂和如何应用系统注入蜜蜂飞行的肌肉的药物。

Watch this Scientific Journal Video about Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera at JoVE.com

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

我们将演示如何实施行为药理学方法，在药物的全身应用（蜜蜂）食欲在蜜蜂的嗅觉空调范式。这种方法允许的内在机制，学习和记忆的形成一个简单而可靠的的方式的调查。

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 ...

behavioural-pharmacology-classical-conditioning-proboscis-extension

Research

JoVE Journal

Neuroscience

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

19.2K 次观看.  Freie Universität Berlin. 我们将演示如何实施行为药理学方法，在药物的全身应用（蜜蜂）食欲在蜜蜂的嗅觉空调范式。这种方法允许的内在机制，学习和记忆的形成一个简单而可靠的的方式的调查。

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

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

科研

神经科学

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

使用传统的空调和心率的方法刺激听觉声学和电刺激对歧视行为的测定

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

小鼠强迫游泳实验

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

悬尾实验

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

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). ...

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

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

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

在两个神经元处理阶段在做人蜜蜂同时长期录音

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

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

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

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

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

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

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

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