肯塔基大学生物系，本科课程和艺术与科学学院办公室支持。

1）引言 Proprioceptors是检测关节位置，方向，速度，和肌肉牵拉的神经元。本体是一个独特的感官的方式，因为proprioceptors interoceptors和刺激感身体内，而不是从外面的世界。 在脊椎动物的系统，它会出现许多的联合和紧张受体是没有必要的检测本体信息总值的。 annulospiral和flowerspray（感觉神经末梢）肌纤维的受体已消融以及振动和麻醉剂的研究显示本体需要两个基本受体组 （Burgess 等人。检讨，1982年）。然而，值得注意的是，有其他受体，如在关节，精细的控制动作收集的冗余信息。像脊椎动物节肢动物挂接附属物。因此，这并不奇怪，为脊椎动物中描述的proprioceptors在节肢动物的肢体和关节。 解剖螃蟹chordotonal机关的安排，使每一个人的神经细胞的分析，根据功能。此外，发展问题可以得到解决，作为动物生长或动物时的再生肢体（1991年，库珀和Govind; Hartman和库珀，1993）。一些螃蟹联合chordotonal机关包含数以百计的初级感觉神经元（库珀，2008），这些神经元在关节的位置和运动的范围分馏监控方面。一个不太复杂的本体系统监测关节运动和立场是小龙虾的腹部肌肉受体机关（MROs）（埃克特，1961a，B;麦卡锡和Macmillan，1995）。小龙虾腹部MROs的机械感受器转导中嵌入肌肉的感觉神经末梢，成为一个分级的受体电位，舒展刺激。当潜在的超过阈值时，动作电位会导致轴突的基地。这是什么定义和穗启动“神经生物学”的网站。在这个系统中的细胞体驻留在密切同位监测的肌肉。存在两个不同类型的牵张感受器在这个感官系统：一个慢慢适应和迅速适应的受体。这次活动是依赖于机械拉伸强度。在小龙虾的MRO系统是类似于哺乳动物的梭内肌梭和肌肉也有传出的控制，保持在哺乳动物梭内肌的肌肉绷紧的性质。 在哺乳动物肌梭的感觉神经元是具有挑战性的electrophysiologically的感觉神经末梢的小性质进行调查，因为。在其周边的结局背根神经节细胞体的位置也很难跟踪。相比较而言，在小龙虾的MRO神经元易于长期录音细胞内外电极。 MRO的感觉神经元的胞体也比较大（直径在50-100微米）。在解决如何“舒展激活”通道在神经元的功能，离子流，渠道分销，和感觉神经元密度（Brown 等 ，1978年的模型， 爱德华兹等人，1981年;埃克斯莱本，1989年还担任感觉神经元;亨特等人 ，1978年Purali和Rydqvist，1992年; Rydqvist和Purali，1991年; Rydqvist和Swerup，1991年，Cooper 等人 ，2003年）。集成在一个段的MRO的感觉输入，可以影响其他毗邻段（埃克特，1961a，b）项。有感觉输入调制从几个报告的MRO（Pasztor和Macmillan，1990年，库珀等人 ，2003年）。调节神经回路是一个丰富 ​​的地区为基础科学的未来调查，可以作为该制剂在哺乳动物为未来的应用奠定了基础，可能在脊椎动物的脊髓（Rossignol等，2001年，2002年; Donnelan ，2009） 1.1）学习成果在这个实验室实验，解剖小龙虾腹部，学习相关的解剖学和生理学的MRO。一是将学习如何监测与神经元的活动外录音，并使用普通的电生理设备。图和解释所获得的数据的基础上所提供的感官刺激。感官刺激，将范围从静态的立场，以及被监视的网段的动态运动。一是将解决这个感官系统的概念及其意义的本体感觉。感觉适应，将在一系列的实验观察。将讨论由学生的意义，以及感官适应背后的潜在机制。 2）方法 2.1）材料<oL&gt; <li>法拉第笼</li><li>微操作机器人</li><li>吸电极</li><li>解剖显微镜</li><li>高强度照明灯（光源） </li><li>显微镜平台</li><li>差分放大器的AC / DC（AM系统公司型号3000） </li><li> PowerLab系统26T（公元仪器） </li><li>头阶段</li><li> LabChart 7（ADI公司的仪器，CO，美国科罗拉多州的斯普林斯，） </li><li>小龙虾盐水（MM：205氯化钠5.3氯化钾13.5氯化钙2 .2 H 2 O; 2.45氯化镁2 .6 H 2 O 5 HEPES调节pH值7.4） </li><li>亚甲蓝：这是小龙虾的盐水浓度为0.25％ </li><li> Sylgard涂层菜（陶氏康宁; SYLGARD 184硅酮弹性体套件，MI美国道康宁公司，美联） </li><li>解剖工具</li><li>昆虫引脚</li></ol> 2.2）安装<img src="/files/ftp_upload/2323/2323fig1.jpg" alt="图1" /> 图1：设备成立<ol><li>安装了法拉第笼。显微镜，高强度照明灯，显微操纵器，盐水浴都将设立内笼（法拉第笼是用来阻止外部电场可能干扰与电气录音）。 </li><li>安装在一个位置，它俯瞰显微镜阶段的显微镜。 </li><li>高强度照明灯在方便的位置的位置。 </li><li>在Sylgard菜准备使用小龙虾的盐水，盐水浴，放置在显微镜下（这是将被放置在解剖小龙虾腹部）。 </li><li>定位在一个位置，吸电极容易获得的盐水浴显微。 </li><li>吸起来生理盐水，直到它与银线内吸电极接触。排列吸电极靠近电极尖端切端线，使两条电线将盐水浴接触。 </li><li>的AC / DC差分放大器（放大器）连接到电源实验室26T。通过适当的线连接，从输入PowerLab系统26T 1到放大器的输出。 <ul><li>放大器的仪器控制应设置以下设置： <ul><li>高通直流</li><li>陷波器关闭</li><li>低通- 20KHZ </li><li>逆时针容量比赛.- </li><li>直流偏移的精细和课程旋钮逆时针</li><li>直流偏置（关） - 关</li><li>增益旋钮- 50 </li><li>输入（DIFF单声道GND） - 差速器</li><li>模式（刺激栅极REC） - 拍摄</li><li> ΩTEST- OFF </li></ul></li></ul></li><li>连接头阶段“输入探头放大器。 </li><li>自吸电极连接的电线头阶段。导线应连接在左上角的红色（阳性），在中间绿色（地），黑（负底部，这是在图2表示。地线可以在盐水浴。 <img src="/files/ftp_upload/2323/2323fig2.jpg" alt="图2" /> 图2：主管级配置</li><li>现在笔记本电脑的USB线连接PowerLab系统26T。确保放大器和PowerLab26T插入并打开前开放的计算机上LabChart7。 </li><li>打开LabChart7。 <ul><li> LabChart欢迎中心中会弹出打开。关闭它。 </li><li>点击安装程序</li><li>点击通道设置。渠道的数量更改为1（盒左下角），按下OK。 </li><li>在图表的左上角设置每秒约2K的周期。伏特（Y轴）设置约500或200mV的。 </li><li>通道1上按一下右边的图表。点击输入放大器。确保设置：单端AC耦合，并反转（反转的信号，如果需要的话），抗混叠，检查。 </li><li>要开始录制按开始。 </li></ul></li></ol> 2.3）夹层<ol><li>小龙虾（Procamarus克氏原螯虾 ）测量体长6-10厘米，应放在冰，麻醉开始前清扫动物。 </li><li>从后面的爪子，用一只手握住麻醉的小龙虾。很快，削减从眼窝中间的头两侧，然后斩首小龙虾（注：从筹备的血液会被粘当它变干，所以洗的工具时完成）。 <img src="/files/ftp_upload/2323/2323fig3.jpg" alt="图3" /> 图3：小龙虾斩首</li><li>一旦小龙虾是斩杀，切之间的胸部和腹部的腹面（尾）。它应该很容易分开的胸部腹部。注：如果小龙虾是男性，削减STYLETS关闭之前分开的腹部和胸部（男性生殖部位）。 （如下图4A所示）。 </li><li><img src="/files/ftp_upload/2323/2323fig4.jpg" alt="图4" /> 图4：腹部隔离</li><li>广场内的腹部，一剪刀的刀片和剪刀的技巧指向远离，沿外侧缘切开的准备。在另一侧重复上述步骤。 <img src="/files/ftp_upload/2323/2323fig5.jpg" alt="图5" /> 图5：纵向剥离腹部</li><li>拿镊子后端（＃3），推的肌肉和胃肠道远离背侧的准备。要确保不推肌肉。 </li><li> （横向外侧）的最后一根肋骨上的剪切删除尾部腹侧。 <img src="/files/ftp_upload/2323/2323fig6.jpg" alt="图6" /> 图6：去除腹部的腹面部分</li><li>出现在小龙虾的盐水（修改后的范Harreveld溶液）的准备工作。 </li><li>准备在显微镜下看，深伸肌内侧肌肉（DEM），可位于一个螺旋扭曲的纤维，和深伸侧肌肉与线性纤维，可以区分（见附件图1和2）。放置两个针在正中矢状地区DEM的肌肉之间的第一和第二肋骨（图7）腹部末端的一部分。 <img src="/files/ftp_upload/2323/2323fig7.jpg" alt="图7" /> 图7：安全准备记录包含维修的感官神经的神经。 </li><li>将用于记录运行的角质层（图7），沿外侧缘旁的神经束。 （这可能是必要的，以打击的准备，或拖放到盐水浴，使用滴管来查找它的运动神经） </li></ol>注意： 每个腹节有两套迅速，慢慢适应左，右hemisegments MROs。相关的神经束沿着外侧缘旁的角质层。这是神经束，将录制。一个将无法查看MROs，因为他们正在位于警报1动作延迟时间和2肌肉（附录图1和2）。图8提供了一个以孤立的腹部解剖概述。 <img src="/files/ftp_upload/2323/2323fig8.jpg" alt="图8" /> 图8：一般夹层概述隔离腹部。 A，B和C是解剖小龙虾的一系列步骤。 3）结果 3.1）记录图9描述了从缓慢和快速适应MROs同时拉伸和维护舒展获得的广义响应。在这次演习中，人会被记录来自MROs在一起，其轴突在相同的神经束中。 <img src="/files/ftp_upload/2323/2323fig9.jpg" alt="图9" /> 图9：小龙虾有两种类型的神经元在MRO 。阶段性的，这是由快速运动轴突和缓慢运动轴突支配进补支配。 （一）当补药受体刺激慢慢适应刺激和动作电位的持续稳定的射击模式。 （b）当一个阶段性的受体刺激迅速适应的刺激，只触发一个动作电位的短格局。 其中包含运动和感觉神经元的神经是整个记录（图10）。但将只检测电机驱动器已被切断腹神经索动物的感觉神经元。 <img src="/files/ftp_upload/2323/2323fig10.jpg" alt="图10" /> 图10：神经束被吸进的记录电极。 （一）免费神经漂浮在解剖腹部。 （二）概述了由神经的神经束和吸塑电极接近。 （三）节段性神经被拉入吸电极，这是在蓝色概述。 一个是现在准备记录MROs的电反应。 <ol><li>清扫范围的筹备工作，并准备录制成立。 </li><li>电地面放置在洗澡和另一端的一个共同点的一个氯化银地线洗澡。注意：有时，这可能会导致在录制过程中的电气噪声。如果发生这种情况并不地面洗澡。 </li><li>使用显微镜，找到要录制的神经 注：寻找最方便的神经段。神经是白色的，可以使用吸管周围神经的盐水喷雾或<html>轻轻吹上的准备。这会导致神经走动，使得它更容易识别。 </li><li>现在的神经已经确定的地方吸电极直接通过神经的显微操纵器（图10）。 </li><li>轻轻拉动注射器上吸引到电极（可以看到被吸入电极显微镜的使用与神经）的神经。 </li><li>按下启动按钮上Labscop​​e7。 </li><li>使用镊子轻轻移动的小龙虾的尾巴，并从180到45和90角度。请注意在每一个不同的角度的录音的区别。 （改变角度的位置时一定要注意与相关的神经元活动在屏幕上的角度变化。，这是可以做到可以使用软件中的注释标记。） </li><li>响应的动作记录在下面的图表不同的静态运动。 <ul><li>保持在45 ° 15秒的小龙虾尾。 </li><li>按下停止LabChart7屏幕上。记录的动作电位的发生，在最后录制的第二。 </li><li>一分钟，保持在45 °的小龙虾尾。 </li><li>按下停止LabChart7屏幕上。记录的动作电位的发生，在最后录制的第二。 </li></ul>说明：实验室图表屏幕上是一个移动的图形，是毫伏（Y轴）与秒（X轴）。措施，在一定的时间发生多少动作电位。它还措施伏的每一个动作电位或细胞外场电位的幅度。振幅是一个相对值，可用于确定是否存在细胞外记录的动作电位的不同大小的。外场电位记录的动作电位被称为“秒杀”。远离神经穗将较小。更宽松的吸电极接头较小的尖峰将作为当前是失去了与周围神经束的密封电阻和低的吸电极开放。 </li></ol>图1： <table border="1"><tbody><tr><td> 角（°） </td><td> ＃1秒3秒后的动作电位</td><td> ＃动作电位在1秒10秒后 </td></tr><tr><td> 180 °（平） </td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td> 45 ° </td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td> 90 ° </td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr></tbody></table>去思考，同时进行这些实验的问题是：是否有图案和一致的反应的延伸和关节的伸屈运动吗？寄托或持有的各种固定位置的尾节引起什么样的反应？是响应一致时反复？ 请仔细观察反应的各种票据。你满意你的观察后，使这项活动的永久记录，保存的数据文件。一旦与你有段3中提出的意见表示满意，移动从4或5段的神经进一步的录音或上段3观察活动的另一侧。 不妨以确定是否神经调质（章鱼胺，羟色胺，proctolin等）或其他化合物或生理盐水离子性质的改变组成定义生理盐水获得的产生不同的反应。 3.2）与亚甲蓝染色人们也许可以解剖出肌肉（见附件），以查看与染色技术MROs。以编制和倒入小龙虾的盐水了。亚甲基蓝溶液约5毫升放入准备，轻轻摇动几分钟菜。然后倒入废液容器过剩亚甲蓝和新鲜的生理盐水倒到准备。现在地方的菜，在显微镜下开始解剖肌肉查看MROs。沿肋骨（外侧正中矢状）剪刀放在肌肉下，拉起你沿着肌肉切，切段。一旦被切断的Del 1和2的肌肉，然后剥离肌肉背部和薄薄的肌肉层（SEM）应遵守。 MROs是最后两个内侧纤维躺在平行螺旋肌（图11）。 “ALT =”图11“/&gt; 图11：腹段的原理说明的肌肉群（A）和蓝色（B）有助于划定的肌肉群，在一个完整的准备与亚甲蓝染色准备。在B在A的概述面积扩大会议的看法。在B，警报1动作延迟时间和2个肌肉群不切掉，在原理图的下半部所示，在A所示对于学生的练习，不妨有学生回答下列问题： <ol><li>什么是interoceptive受体？ </li><li>如何interoceptive受体与proprioceptors？ </li><li>解释感觉适应和如何它可能涉及到这个实验室。 </li><li>什么是范围分馏？是什么使中枢神经系统做呢？ </li><li>录得阶段性或强直型神经元的神经元？为什么呢？ （提示：你会怎么假定运动神经元的活动看起来感觉神经元活动像在一个完整的动物） </li><li>在另一张纸上画一个小龙虾尾角与AP的频率图。 </li><li>解释任何图中的趋势，你可能会观察</li></ol>

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为了充分记录在小龙虾的MRO 活动，在原地，在提供相关的电影和文字的细节提供了关键的步骤。我们的报告的目标之一是增加在该制剂的潜在的意识在学生调查实验室教授在感官生理学的基本概念。的筹备工作中的生存能力非常强大的，而正沐浴在最小的生理盐水。 电机控制的MRO肌肉已被确定，但在传输和突触可塑性以及潜力的兴奋和抑制性神经元的传输效果的调节仍然是一个开放的调查面积（Elekes和弗洛里，1987A，B，弗洛里和1955年，弗洛里; Kuffler，1954年; Kuffler和Eyzaguirre，1955年）。 该制剂可用于研究的实验条件下的数量以及范围内动物的自然运动，以便更好地了解生物学的主要研究以及示范目的的MRO。这些感觉神经元的生物物理特性已部分得到解决，他们适应的性质，在神经活动与保持刺激（Brown等，1978年，爱德华兹等人，1981年; Purali，1997年; Rydqvist和Purali，1991年; Rydqvist Swerup，1991年）。然而，只有少数报告对这些感受器的地址和相关的肌纤维 （Cooper等人，2003年; Pasztor和Macmillan，1990）神经调节。只有少数的许多化合物是已知的血淋巴中的报告处理。许多调制器和调制鸡尾酒仍然要复杂的MRO（肌肉和神经元）检查。 Pasztor和麦克米伦（1990年）审查各甲壳类MROs的活动5 - 羟色胺和章鱼胺的神经调质，并指出，有不同物种。他们并没有详细研究这些神经调质的长期影响，也不对活动的影响，在不同的静态位置的MRO。 这种类型的准备，可以帮助了解感官知觉和调节神经处理，这是在人类与运动单位abnormities（Patel等人，2009年的康复和疾病管理的重要基础;。拉宾等人，2009年 ，圣马力诺等。 ，2010）。可用于输入和监测在访问无脊椎动物的准备关节运动的射击模式的各类机器人/假体（麦克米伦和Patullo，2001年）。还有许多问题，等候在此准备的答案，在许多方面可以。

<ol>
<li><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank">PowerLab 26T (AD Instruments)</a> </li>
<li><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 7 (ADI Instruments, Colorado Springs, CO, USA)</a> </li>
</ol>

muscle receptor organs in the crayfish abdomen: a student laboratory exercise in proprioception

本实验的主要目的是为了证明初级感觉神经元关节运动和岗位作为一种动物的本体信息传达的信息。这个实验的目的是学习准备的解剖结构，在解剖显微镜下染色，解剖和观察神经元和感觉结构。这是使用基本的神经电生理设备记录电活动的联合受体器官和染色技术。在小龙虾的肌肉受体的器官（MRO）系统是类似于哺乳动物梭内肌梭，艾滋病作为一个比较的模型，更易于访问的电生理记录。此外，这些都是识别的感觉神经元之间的准备工作。准备是可行的，这是对学生实验练习适合在一个小时的最小生理盐水。 MRO为也易患神经调节，鼓励行动的调节和整合运动和动态信号与系统中的一个可以改变增益静态位置的网站有趣的问题。

Watch this Scientific Journal Video about Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception at JoVE.com

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

本实验的主要目的是了解如何初级感觉神经元关节运动和岗位作为一种动物的本体信息传达的信息。本报告的另一个目标是目前解剖解剖，在解剖显微镜下观看的神经元的准备。

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 ...

muscle-receptor-organs-crayfish-abdomen-student-laboratory-exercise

Research

JoVE Journal

Neuroscience

22.1K 次观看.  University of Kentucky. 本实验的主要目的是了解如何初级感觉神经元关节运动和岗位作为一种动物的本体信息传达的信息。本报告的另一个目标是目前解剖解剖，在解剖显微镜下观看的神经元的准备。

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

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

小龙虾的反思记录行为反应

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

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 ...

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

小龙虾腿伸肌高和低输出NMJs生理录音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

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 ...

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 ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

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 ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...