作者感谢Hyewon库珀的艺术贡献。

1。解剖和记录电活动从Propodite-dactylopodite（PD）的神经<ol><li>从后面握住整个蟹头胸甲，并避免爪，捏钳的meropodite的近端部分。腿会autotomize以防止动物流血过多而死。处理蓝蟹的时候，因为他们是相当积极的，非常快，请小心。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig1.jpg" width="500px" /> 图1，首先走了蟹的腿 。 chordotonal器官（阴影区域）的解剖位置叠加在这个示意图。 。双箭头表示从哪里横切腿的PD神经实验<a href="https://www.jove.com/files/ftp_upload/51050/51050fig1highres.jpg" target="_blank">点此查看大图</a> 。 </li><li>使propodite和carpodite之间的切割。丢弃carpopodite和t<html>他重视meropodite（ 图1）。 </li><li>削减与一个＃11刀片（ 图2和图3）注意手术刀的propodite的色素（横向）边一个大窗口，角质层：不要切深。 </li><li>由下方滑动的手术刀刀片拆除角质层和平行于角质层。这切断附着在角质层的肌肉纤维。 </li><li>使用相同的技术削减了pigmentless的propodite的（内侧）一侧较小的窗口，但保留髁（插座接头或分部之间的铰链）连接完好。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig2.jpg" width="500px" /> 图2沿上propodite虚线剪下 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 N“SRC =”/ files/ftp_upload/51050/51050fig3.jpg“宽度=”500px的“/&gt; 图3：暴露在窗口中的神经（箭头勾勒 ​​出神经束）。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>制备含蟹生理盐水断引脚制备的Sylgard林立盘注：参看表1种特定盐水食谱。 </li><li>通过与火抛光玻璃针小心地探测定位在PD的器官。的弹性绞合线，横跨关节具有银色外观。 </li><li>除去肌纤维，来自腱的两侧遮住了器官的视图。要非常小心，不要伤到PD的机关及其神经。 </li><li>一旦这项工作完成，重新连接牢固编制朝上（ 图4）的菜与颜料侧（外侧）。 <img alt="图4" fo:content-width=“5英寸”SRC =“/ files/ftp_upload/51050/51050fig4.jpg”宽度=“500px的”/&gt; 图4。外露的PD器官和神经 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>遵循propodite PD的器官如神经近端远越好，以腾出一长段神经（1.5厘米）记录的目的。这是最好的做法，而PD的神经仍连接到主腿部神经。 </li><li>用玻璃针的帮助下分离主腿部神经PD的神经后，近端切断PD的神经与虹膜剪注意：在解剖过程中，不要拉长或拉的神经。 </li><li>移动dactylopodite来扩展和完全屈曲位。注意到关于那里的极端屈伸位置是和一个中间点，供以后使用。 </li><li>将接地线盐水浴内。 </li><li>打开电子lectrophysiology记录硬件/软件注：我们的设置以前已经23描述。 </li><li>放置显微镜，使其俯瞰显微镜载物台。一旦准备盘放置在舞台上，你将需要调整的高强度照明光束的位置，以最佳的可视化的准备。 </li><li>定位显微操纵器，以便连接的吸电极组件将很容易获得的盐水浴和准备。抽吸电极的构造如图在线视频24。 </li><li>以检测神经活动，画出PD神经的切断端插入抽吸电极。 </li><li>将长短格整个几个周期与玻璃探头或木榫的帮助扩展和弯曲位置。 </li><li>接着观察活动时的长短格被固定在延长，弯曲，和中间的位置。 </li></ol> 2。从张力记录电活动神经同时监测部队产生<ol><li>确定腿的外侧和内侧。内侧具有柔软的手感，可以通过用指甲轻轻夹持在meropodite区域被感觉到。 </li><li>把这个柔软的角质层一面朝上的菜。该励磁电机的神经支配的肌肉的揭幕战也支配的carpopodite担架肌肉。 </li><li>为了刺激开瓶器肌肉的拉伸运动神经在carpodite区域分离并与抽吸电极刺激。腿的近端部分被横切meropodite用剪刀除去。 </li><li>去除角质层中的腕区域的部分的内侧（内侧， 图5A）。 </li><li>切弯肌肉的apodeme并小心地取出肌肉不拉主力腿神经出腿腔（ 图5B，C注意箭头在哪里德尔apodeme分离）。主要的腿神经和胸罩NCH ​​到担架肌肉然后可以观察到（ 图5D）。 </li><li>找到神经从主神经束分支到担架肌肉（ 图5E中，在箭头所示），这可切割靠近肌肉和拉入抽吸电极刺激（ 图5F和G，箭头示出了分支）。 </li><li>梳理出神经的部分，伸向了​​开门红，而不会挤压神经。横切担架/首战运动神经。 </li><li>拉运动神经进入抽吸电极，然后刺激它。 </li><li>揭露首战肌肉和紧张的神经去除接近肌和propodite的腹侧区域。切断与一个＃11刀片在propodite（ 图6）手术刀越接近肌肉注：不要深深地砍在近端区域，因为它可能会损坏首战运动神经。 <img alt="图5" fo:content-widtH =“5英寸”SRC =“/ files/ftp_upload/51050/51050fig5.jpg”宽度=“500px的”/&gt; 图5。解剖步骤露出运动神经元对刺激的揭幕战肌肉 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig6.jpg" width="500px" /> 图6。削减角质层的propodite，露出接近的肌肉，因此它可以被删除的腹侧半 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig6highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>换用新鲜冷却液的生理盐水整个解剖过程中，以保持神经元的存活。如需进一步解剖，将准备的菜在解剖显微镜下，用光纤照明。 </li><li>小心地从它的附件削减接近肌腱使用尖锐中型剪刀长短格。 </li><li>要非常小心，不要从主腿部神经应该是清晰可见的打扰跳转到揭幕战的肌肉。 </li><li>取下并扔掉接近肌肉和肌腱。 </li><li>使在长短格一个孔用解剖针。该孔将在后面用来勾上的张力（力）换能器的金属销，但有必要使其在这一阶段的程序。 </li><li>找到神经分支投射到揭幕战肌肉和apodeme在揭幕战中肌肉的远端区域。仔细探查神经与火抛光玻璃的工具。 </li><li>观察神经张力所产生的apodeme并且前进到运动神经束的前端。 </li><li>为了检测神经活动，填补吸电极与蟹生理盐水并绘制揭幕战的紧张神经的切断端插入电极。确保吸入电极紧密配合的神经。 </li><li>检查为神经氨酸升被动张力开门红apodeme的相关性。同时记录电活动从紧张的神经，迅速 ​​旋转长短格成一个扩展接头（ 即拉伸开门红肌肉）。 </li><li>接下来，测试的积极力量发展有关的刺激频率。 </li><li>牢固附着的金属挂钩，这样的梭尖穿过一个小孔长短格。 </li><li> ，附加的金属钩的另一端连接到力传感器注意：确保换能器在一个90°角的每个时间，使该引脚为垂直于传感器的最大检测所产生的力。 </li><li>拉动钩和长短格，以便它在约45°的开瓶器关节的角度。 </li><li>现在，刺激运动神经，在100赫兹为250毫秒，测量力和张力神经的发射频率。 </li><li>将联合到一个完全伸出的位置，使得开启者的肌肉纤维松弛。 </li><li> Stimulate运动神经在100 Hz时250毫秒，测量力和张力神经的发射频率。 </li><li>弯/弯曲的关节，以便它完全弯曲（〜90°）。在这个位置上揭幕战的肌肉纤维得到充分拉伸。有可能是由换能器由于这种被动拉伸肌肉的测量一些力。 </li><li>刺激运动神经，在100赫兹为250毫秒，测量力和张力神经的发射频率。 </li><li>测量力后，进行了一系列不同频率的：20，40，60，80，100赫兹为8-10秒，每个关节的位置。 </li><li>衡量一个快速释放紧张的反应。安排的制备使得在力传感器的钩子可以容易地推压在长短格孔出来。 </li><li>弯/弯曲的关节，以便它完全弯曲（〜90°）。现在，刺激运动神经，在100赫兹，5秒的持续刺激。 </li><li>第一次生后d以下，由于工作紧张已建立了上揭幕战的肌肉，推动销出的长短格孔。 </li><li>之前和之后的针保持的长短格的释放研究神经张力的记录。 </li><li>的神经活性物质，如章鱼胺，五羟色胺，和proctolin，这改变了张力的神经元的输出的调节作用，可以通过添加这些分子以入浴介质在暴露的开瓶器肌肉和重复实验来确定。 </li><li>用移液管和在准备滴1-2毫升。 </li></ol> 3。染色外周神经系统结构的甲壳步足<ol><li> 亚甲蓝技术 <ol><li>稀释1部分的亚甲基蓝氯化物溶液（0.25％）与蒸馏水两部分。 </li><li>加入此混合物中，以5份缓冲盐水。彻底冲洗感兴趣的区域。 </li><li>根据第1条的协议剖析了一条腿。孵育前paration中的亚甲基蓝溶液在12-13°C。 </li><li>检查制剂每10-15分钟，用低强度照射，以遵循染色的进度解剖显微镜。在某些情况下，染色会在一小时内完成，在别人可能需要过夜。 </li></ol></li><li> 4 -二-2 - ASP技术 <ol><li>荧光染料可用于回填的帕金森病的神经元，或者直接从浴中染色的制备。然而合适的显微镜的能力，以查看该荧光染料是必需的。 </li><li>根据第1条的协议剖析了一条腿。 </li><li>孵育的制备在4 - 二-2 - ASP溶液10μM的浓度，并使制剂在冰箱中15分钟。使用适量的解决方案涵盖了准备。 </li><li>快速拍摄的准备工作，避免过度暴露于汞的光。这种荧光染料消失相对较快。 </li></ol></li><li&gt; 氯化钴技术 <ol><li>根据第1条的协议暴露PD的神经，并保持它浸在冷盐水。 </li><li>使凡士林以及持有氯化钴。如果有任何氯化钴泄漏到盐水浴的全部准备工作将弄脏的黑色，并准备应该被丢弃。 </li><li>滑的聚苯乙烯片材的一个小切口，以使一个塑料平台，并针它以这样一种方式，它不会漂走或成为浸渍在盐水浴（ 图7）。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig7.jpg" width="500px" /> 图7。聚苯乙烯板材用大头针在地方举行的菜 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig7highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>从细皮下注射针头固定到一次性注射器喷出凡士林，然后做一个屏障（一个圆可能会很好地工作），这应该是大约1-1.5毫米高，除去浅“V”在中点所在的神经会被披上跨越，对聚苯乙烯的滑之上。 </li><li>使盐水对附近的“V”形的屏障两侧的小水坑。注意不要拉伸或挤压神经，从盘中仔细解除神经，并将其放置在石油生理盐水水坑果冻很好。 </li><li>工作迅速，使神经节不干涸，仔细弹出凡士林，以覆盖外露的神经。现在测试用生理盐水障碍，并确保它不被泄漏。 </li><li>吸干了盐水在闸厢内侧，看看它填补了洗澡的时候生理盐水是围绕高凡士林的墙上，以确保双方彼此（ 图8）隔离。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig8.jpg" width="500px" /> <stro<html> NG&gt;图8。 凡士林很好用生理盐水和PD神经跨越好 。 PD的神经还没有被切断呢。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig8highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>吸走生理盐水中使用的是一块纸巾井。避免让纸包裹的神经。 </li><li>创建一个新的水坑运用的几个小滴蒸馏水，然后切神经末梢。要非常小心，不要穿过屏障使切口拉出时神经。蒸出的水的渗透休克将“气球”的轴突。 </li><li>在30秒内加一小滴氯化钴的水中，浸泡了该解决方案，然后添加足够的氯化钴，形成一个水坑在神经（ 图9），这缩短的部分。准备最好保持冷藏在13℃下12-24小时。 tp_upload/51050/51050fig9.jpg“宽度=”500px的“/&gt; 图9。切割PD的神经暴露于氯化钴 。切断神经肿起来的水的存在，通过轴突成神经元细胞体推动并加快了染料分子的运动。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig9highres.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>拆下加湿水坑。吸干了使用组织的钴溶液洗去钴的残余盐水的一些变化。 </li><li>隔离准备转移到含约10ml生理盐水蟹的小玻璃培养皿。的神经元进行洗涤，以下步骤在原位进行。良好的金属工具不被用于处理在此步骤之后的制备（应使用特定的工具，并不在稍后的时间为生理学被使用）。 </li><li>加1-2滴硫化铵（NH4）2 S的盐水。盖上（NH 4）2 S瓶紧紧地放置回罩。观察解剖显微镜下的准备反应</li><li>在1-2分钟，钴充满神经元及其过程应该开始出现，因为它们会弄脏的黑色。 </li><li>经过5-10分钟，更换新鲜生理盐水开发解决方案。 </li><li>请确定您已经倒入水槽排水的开发解决方案，其次是流动的自来水几分钟或浪费，瓶子，盖子。 </li><li>倒出盐水和修复神经准备用Bouin氏液固定剂两种变化约15分钟。对于像揭幕战肌肉组织较大（染色紧张的神经元），增加固定的持续时间30分钟。 </li><li>脱水在一系列乙醇浓度开始在70％（ 即 70％，80％，90％，100％）按升序排列。约10分钟，在各浓度足以小的组织。 </li><li>后在100％乙醇的两个变化约10-15分钟，清除组织了100％水杨酸甲酯代替乙醇。准备将留在这个解决方案为永久重复观看。随着时间的填充单元将变得更加明显，因为周围组织将变得更加清晰。强化方法可以用来帮助防止褪色随着时间的推移25。 </li><li>使用相同的过程，填补了紧张的神经与氯化钴。然而，改变乙醇溶液多次在每个乙醇脱水步骤，并培育内酒精的准备工作约20分钟每步彻底脱水的肌肉，让他们清楚好。 </li></ol></li></ol>

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作者宣称，他们有没有竞争的财务权益。

这组实验的目的是1）从可识别的本体感受器官和神经紧张教导，并表现出细胞外记录的基本原则和2）强调有关特定感官系统的生理功能解剖标测的重要性。这种实验方法及动物模型使用的是价格便宜，比较容易开展神经生理学教学实验室。 chordotonal器官的神经元有两个特定的功能类型，那些对运动作出反应和回应那些对静态位置。从各种chordotonal器官的单个细胞记录，无论哪个关节进行检查，都显示出这是事实3,5。事实上，随着龙虾的触角关节相关chordotonal器官揭示两个相同的感官类型和基本解剖学27。除了有作为两个神经元类型（移动，positi上）时，神经元共享它们各自的弹性股线的同一解剖结构。大胞体位于近端上链往往属于动态运动敏感神经元。该信号静态持仓神经元有小胞体，位于远端。这些细胞是tonically活性。 PD的合资只包含一个单一的chordotonal器官，同时也有在腕骨-propodus（CP）和merus-腕骨（MC）接头2 chordotonal器官。 揭露本体结构蓝蟹（C.蓝蟹 ）的电生理记录解剖需要一个战略，让关节活动，以发生在自然的位置，而从感觉神经元记录。紧张的神经在行走的腿肌肉的揭幕战是一个非常精细的神经由几个神经元。除非小心，张力神经以及运动神经支配的肌肉被刺激，这可剥离时被损坏。为最佳录音抽吸电极需要进行调整以适应神经的大小。录音是在使用30-40X的放大倍率解剖显微镜和低端显微操作一个实验室的学生容易获得。 未来的实验，这将是有趣的，寻求与联合chordotonal机关将在生命周期的不同阶段来考察过程中不同物种的腿再生的结构和生理型材的后续行动所使用癌症魔导师 19,26的初步研究。剩下要解决的问题是：1）没有再生肢体再生时，神经元的分布和组织取决于动物的年龄，2）是一个再生肢体功能的轴突投射到中枢神经系统（腹神经索）还是它需要时间和联合使用来建立连接的功能，和3）会发生什么，以切断的轴突近端自残平面时，肢体autotomized？28 甲壳类动物顺应环境条件和其周围的温度，但目前还不清楚它们如何保持协调内的神经电路的神经元改变它们的活性响应于温度的变化。变化的速度慢可能让动物有一段时间的适应，而快速变化可能not29，30。由于代谢，行为31，或者环境影响生理变化，pH或渗透压可能存在类似的挑战，以参与本体感觉神经回路。这些甲壳类制剂是理想的解决这些类型的问题，因为它们的功能被很好地表征在单细胞水平。 在这个协议中，我们已经证明紧张的神经元在监测的揭幕战肌肉产生力量的生理意义。这些张力感受器可以通过染色程序追溯到apodeme中的位置。这些神经元，在哺乳动物中，检测力在各层次，增聘神经元的力增大。在活动的频率是关系到运动神经元的刺激频率，直到饱和接收为止。使用带有弯曲的指节关节快速释放协议，紧张的活动很快消失，但随后在后完全伸展关节恢复张力返回。这是一个典型的实验程序，以说明张力感受器测量的力。各种神经调质可应用于编制，看看它是如何影响力和神经反应的发展。其中一个重要方面是如何的神经反应处理和集成在中枢神经系统和其上运动神经元活动的影响。我们已经证明的技术允许一个开始，以解决有关的张力（感觉）神经运动神经元电路的功能，在一个完整腿的神经节，即信号和背部的更多信息到肌肉。 展示了染色程序，关键是理解的感觉神经元的生理该支配本体感受器官。基于功能与胞体大小的神经元的解剖安排在蟹腿内的各种chordotonal器官相似。它不知道，如果类似神经元的安排也存在于其他甲壳类动物或昆虫。从单细胞结合生理记录和绘制的位置可以直接结构与功能的关系。长期保存用的CoCl 2染色和固定的解剖结构允许一个反复使测量和评估的结构布置。 本体感觉和骨骼肌的紧张前台的感官方式，使协调的行为和应对外部和内部环境进行阐述动物在各种骨骼肌配置秒。在小龙虾的腹部肌肉受体的器官是另一个证据充分的准备（见小龙虾项目; <a href="http://www.crawdad.cornell.edu/">http://www.crawdad.cornell.edu/</a> ）作教学用途本体的每腹部半段23只有两个神经元。如果能够从单个神经元记录到感觉神经束提供进一步的细节了解的感官接收的基本原理，援助。这些相对简单的甲壳类动物制品允许一个解决本体感觉和紧张监控的基本方面，有可能确定神经回路，使之本体感觉和其他感觉输入9-12，32，33中央集成。

本体是肢体位置和运动，使协调运动行为的感觉。本体感受器包括位置（静态）和运动（动觉）受体。在昆虫和甲壳类动物，chordotonal机关是这些信息提供给中枢神经系统1的结构。不是所有的chordotonal器官跨越关节，但他们仍然可以监视关节运动，由于其附着在其上跨越关节和移动与骨骼肌肉和关节的关节协会的apodemes（腱状结构）。蟹腿有6节，每一个都具有一个或两个chordotonal器官2。通常情况下，chordotonal器官有60-100或多项嵌入弹性链内的感觉神经元，即信号静态关节的位置，方向和运动3-6速度神经元。从chordotonal器官输入的每个关节和腿部，然后集中处理，使协调运动的动物。 下面展示了程序启用支配这两种类型的受体相对于它们的位置上的chordotonal弹性绞合线和apodeme神经元的结构和功能的分析。为了说明这一点，我们使用propodite-dactylopodite（PD）chordotonal器官，跨越蟹腿3的前端最段的器官。虽然详细的电生理研究开始于20世纪30年代至今仍在进行的，某些方面已成为闻名关于本体感受器中的各个关节和他们的角色在muscles10-16的协调控制段连接。建立本体感受器官，肌肉和神经系统的结构与功能的关系，将有助于进一步定义这些角色。例如，标签的胞体和插入apodeme紧张的神经元末梢的结局，就会发现它们的相对位置的肌肉纤维8,17-21。 我们提出3种染色技术的甲壳类动物的腿，可以在研究或学术实验室中使用。亚甲基蓝染色提供最佳对比度的肌肉和神经，并建议作为一个简单的技术，让学生学习解剖学。具有荧光显微镜实验室的设置可以通过简单地暴露神经完成更多的选择性神经元染色s到的活体染料4 - 二-2 - ASP。第三种方法是氯化钴回填，其染色和固定的神经元，并且不需要荧光成像。虽然它是时间和劳力密集型的，这种染色工艺使高对比度和特异性，将填充的神经。一起这些技术可用于比较各种chordotonal器官，而不是只在一个肢体或肢体之间，而且在其他甲壳类动物和昆虫物种20-22。在生理记录和解剖染色使用的蓝蟹（C.蓝蟹 ）都是现成的四周，美国的南部和东南部边境。本种作为在大多数的螃蟹发现chordotonal和紧张的神经安排的代表。西海岸实验室将倾向于使用更大的珍宝蟹（ 巨蟹座魔导师 ）这些实验。

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			<td height="21" style="height:21px;width:216px;">Sylgard</td>
			<td style="width:184px;">Dow Corning</td>
			<td style="width:119px;">182 silicone kit</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5670</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES acid</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">3375</td>
			<td style="width:167px;">Free acid, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES base</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-Glucose</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">G7021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO4&#8226;7H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2SO4&#8226;10H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">246980</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bouin&#8217;s solution fixative</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">HT10-1-32</td>
			<td style="width:167px;">Caution: Hazardous material (Special shipping cost required)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">CoCl2</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Caution: Hazardous material. Please follow proper disposal according to local and federal regulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methylene blue chloride</td>
			<td style="width:184px;">Matheson Co., Inc</td>
			<td style="width:119px;">Basic Blue 9, C.I. 52015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4-Di-2-ASP</td>
			<td style="width:184px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">4-(4-diethylaminostyryl)-N-methylpyridinium iodide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To chloride silver wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">H1758</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Materials</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting tools</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">assortment</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Intracellular electrode probe</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insect Pins</td>
			<td style="width:184px;">Fine Science Tools, Inc</td>
			<td style="width:119px;">26001-60</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope (100X)</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiber optic lamp</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Small adjustable mirror</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To direct light beam toward the preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass electrodes</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS7095B5X</td>
			<td style="width:167px;">Box of 200, suction electrodes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micromanipulator</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">MD4-M3-R</td>
			<td style="width:167px;">Can fix for base or on a metal rod</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Raised preparation stand</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver wire (10/1,000 inch)</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">782500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Computer</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AC/DC differential amplifier</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">Model 3000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PowerLab 26T</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">27T</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Force transducer</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">0-50g</td>
			<td style="width:167px;">MLTF050/ST</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Head stage</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Comes with AC/DC amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabChart7</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrical leads</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass tools</td>
			<td style="width:184px;">Make yourself</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For manipulating nerves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable and connectors</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipettes with bulbs</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7</td>
			<td style="width:167px;">Box of 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beakers</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wax or modeling clay</td>
			<td style="width:184px;">Any company or local stores</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stimulator</td>
			<td style="width:184px;">Grass Instruments</td>
			<td style="width:119px;">SD9 or S88</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="84" rowspan="2" style="height:84px;width:216px;">Plastic tip for suction electrode</td>
			<td rowspan="2" style="width:184px;">Local hardware store (Watt&#8217;s brand)</td>
			<td rowspan="2" style="width:119px;">&#188; inch OD x 0.170 inch ID</td>
			<td style="width:167px;">Cut in small pieces. Pull out over a flame and cut back the tip to the correct size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;"></td>
		</tr>
	</tbody>
</table>

proprioception and tension receptors in crab limbs: student laboratory exercises

这些程序的主要目的是展示用于教学和研究的目的如何记录生活负责本体初级感觉神经元，因为他们检测关节位置和运动，肌肉紧张的活动。从甲壳类动物本体感受器和张力感受器的电活动记录由基础神经生理学仪器和换能器，用于同时测量，是由刺激运动神经产生的力。此外，我们将演示如何染色的神经元的解剖结构的快速评估或永久固定。染色显示解剖组织，是代表大多数甲壳类chordotonal器官。比较紧张的神经反应，本体感受反应是一种有效的教学工具，在确定这些感觉神经元是如何定义的功能和解剖结构是如何关联的功能。三种染色技术都使研究人员和教师选择的方法，非常适合他们的实验室。

当PD器官被充分延伸的接头伸出，如图所示为图10中的第一，第二运动过程中在帕金森病的神经活动的鲁棒性。某些活性保持，同时它仍保持在打开位置。这一活动是从静态位置敏感的神经元（ 图10中所示的记录的第二半部分）。运动位移过程中唤起的响应，并在烧成过程中chordotonal链的拉伸（ 图11）主要存在。 尖峰的进一步分析可以通过排序的相对幅度可以容易地接近。这是演示的感觉神经元被招募的职位或类型的运动5的不同人群的方法。典型的振幅范围为0.25-1.5毫伏，但这些值是依赖于抽吸电极密封的阻力（ 即气密性）。在T尖峰频率他的各种尺寸范围也可以图形方式表示的分析。 由开瓶器肌肉相对于所述刺激频率产生的力可以通过重叠各自的电压-时间轨迹上彼此（ 图13A和B）的顶部进行比较。这也可以被用于在每个给定的刺激频率的每个关节的位置进行。张力神经的活动然后可以在每个刺激频率产生的相对力的大小相关的，并且对于每个关节的位置。如在帕金森病的神经，各种尖峰振幅被看作响应于所述开瓶器肌肉（ 图13C）的收缩。 在行走的腿神经元的解剖结构被明显地观察到亚甲基蓝染色（ 图14）。注意弹性chordotonal链和紧张的神经元是接近apodeme。几种胞体不同直径的ð特定位置也可见于这个数字。张力神经和担架运动神经的整个过程示于图15。帕金森病的神经个别神经元用4 -二-2 - ASP（ 图16）和氯化钴（ 图17）回填技术显示具有较高的对比度。在高放大倍率的感觉神经末梢可支持性scolopales（ 图16B）21,22,26内可见。 <img alt="图10" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig10.jpg" width="500px" /> 图10，移动并保持在0℃。动态神经元强劲射击运动过程中和静电敏感的神经尖峰存在，而联合举行开放。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig10highres.jpg" target="_blank">点击这里查看大图&lt;</a>/ A&gt;。 <img alt="图11" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig11.jpg" width="500px" /> 图11。快速打开和关闭从完全弯曲到扩展（90-0°）的位置 。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig11highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图12" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig12.jpg" width="500px" /> 从PD神经记录图12。胞尖峰 。关节完全伸直，然后迅速移动到一个半屈曲位，然后保持静止。在运动过程中注意的活性和活性降低静态时。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig12highres.jpg" target="_blank">点击这里查看大伊马</a>GE。 <img alt="图13" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig13.jpg" width="500px" /> 图13。所开发的关节的相对力量充分弯曲，并在不同频率刺激（A）从力传感器电压-时间的痕迹，只显示每个刺激频率。 （B）在A组的痕迹叠加不同的颜色，便于比较。 （C）电压时的电活动的痕迹从紧张的神经在录制时运动神经在80赫兹刺激。注意的刺激伪影相比，神经活动的规则图案。另外，还要注意的神经反应的各种幅度。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig13highres.jpg" target="_blank">点击这里查看大图</a> 。 图行走的腿准备14。亚甲基蓝染色 。个人胞体显示与他们的感觉神经末梢伸入弹性绞合线。接近紧张的神经元会显示在apodeme。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig14highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图15" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig15.jpg" width="500px" /> 图15。从远端（红色箭头）所产生，并加入运动神经（绿色箭头）的紧张神经。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig15highres.jpg" target="_blank">点击这里查看大图</a> 。 图16（A）回填癌症魔导师 PD的神经与4 -二-2-ASP（B）高倍感觉神经末梢的。 <a href="https://www.jove.com/files/ftp_upload/51050/51050fig16highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图17" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig17.jpg" width="500px" /> 图17。神经细胞充满了氯化钴和处理是（A）。所示的染色制剂追踪轮廓（B） <a href="https://www.jove.com/files/ftp_upload/51050/51050fig17highres.jpg" target="_blank">点击此处查看大图</a> 。 <table border="0" cellpadding="0" cellspacING =“0”宽度=“392”&gt; <colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="21" style="height:21px;width:235px;"> 盐水 </td><td style="width:157px;"> g / L的 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;">氯化钠</td><td style="width:157px;"> 27.29 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;">氯化钾</td><td style="width:157px;"> 0.81 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;">硫酸镁4•7H 2 O </td><td style="width:157px;"> 4.81 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;">氯化钙2•2H 2 O </td><td style="width:157px;"> 1.85 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;">的Na 2 SO 4•10H 2 O </td><td style="width:157px;"> 0.97 </td></tr><tr height=“20”&gt; <td height="20" style="height:20px;width:235px;">右旋糖（D-葡萄糖） </td><td style="width:157px;"> 1.982 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> HEPES酸</td><td style="width:157px;"> 0.476 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> HEPES盐</td><td style="width:157px;"> 2.08 </td></tr><tr height="40"><td height="40" style="height:40px;width:235px;">调节pH至8.1，用NaOH或HCl </td><td style="width:157px;"></td></tr></tbody></table> 表1。食谱螃蟹生理盐水。

Watch this Scientific Journal Video about Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises at JoVE.com

Proprioception and Tension Receptors in Crab Limbs | Biology | Video

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

生理和解剖技术演示，以解决功能和结构的关节本体感受器和肌张力受体在甲壳类动物行走的四肢。

本体感觉和张力感受器在蟹四肢：学生实验室练习

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

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25.0K 次观看.  University of Kentucky. 生理和解剖技术演示，以解决功能和结构的关节本体感受器和肌张力受体在甲壳类动物行走的四肢。 

视频: Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学：

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

科研

神经科学

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

膜电位，突触反应，神经回路，神经调节和肌肉组织学用小龙虾：学生实验室练习

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

小龙虾腹部肌肉受体机关：在本体的学生实验室练习

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

生理实验与小龙虾肠：学生实验室的工作

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

测定在异种细胞表面的化学感应受体的表达

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

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 ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...