我们感谢社论援助先生克雷格D.斯拉文（英语系，肯塔基大学）。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）导言运动神经元沟通的一个统称作为一种神经肌肉接头（NMJ）突触的肌纤维。 NMJs可以很容易被访问，在大多数的小龙虾肌肉的准备。许多小龙虾NMJs证明非扣球兴奋性突触后电位（EPSP）的突触后树突内产生的哺乳动物的中枢神经系统或阈下的反应，在脊椎动物NMJs中指出的分级电信号（Wiersma范Harreveld，1938年，卡茨Kuffler，1946年） 。小龙虾NMJs可以作为基本突触模型的突触传递和突触分化成提供一般的见解。 一般来说，运动单位，规范动物行为的各个方面，通过突触的沟通NMJs和肌肉的属性类型。自首次观察到的螃蟹和小龙虾接近肌肉（卢卡斯，1907年，1917年）的“快速”和“慢”的肌肉收缩，类似的肌肉收缩分化已在其他小龙虾的肌肉类型，如腹部屈肌（肯尼迪和武田，1965a ，b）和肢体伸展（范Harreveld Wiersma，1936年）。 “快”收缩启动快速反应。例如，小龙虾尾翻转是一个快速的行为。 “慢”的收缩，保持动作缓慢，并帮助保持姿势（Bradacs等，1997） 。已广泛用于通讯以“快”和“慢”的肌肉收缩，“阶段性/高输出”和“补药/低产出”来形容运动神经元。在速度和肌肉收缩的时间不同的是在与突触前，在突触的结构和突触强度的差异（王等，1996 ）的一部分。肌原纤维蛋白异构体的表达是收缩的差异也很重要，但像腿伸肌，在一个特定的纤维是由两种类型的运动神经元支配的筹备工作，重点是对突触的终端差异，因为终端的份额相同的靶细胞（Mykles等，2002） 。早先的研究中研究的腿伸了两个兴奋的运动轴突和描述的阶段性和补品的表型 （Bradacs等， 1997）。在这份报告中，我们将演示如何进行清扫和取得的录音，以便其他人可以进一步探讨这些神经末梢的突触分化的属性。 观看与透射电子显微镜，各种系列的小龙虾腿伸肌肉的补品和阶段性终端获得的部分透露，补虚终端包含更多的定价比阶段性终端水泡;线粒体中较为多见，在进补的神经元，并阶段性终端的突触更复杂的比那些低产出突触，因为它们包含多个活动区，具有不同间距“（Miller等，2002年，斯通等，2008; King等，1996;。Bradacs等，1997 ）。低输出进补终端也阶段性终端（Cooper 等人 ，2003年）相比，更容易加强与突触传递神经调节血清素（5 -羟色胺） 。 事实上，补品和阶段性NMJ相同的肌纤维上，可以更容易地评估对一个给定的肌纤维突触前的差异，肌肉疲劳，抑郁症的突触和突触的串扰问题。各种问题仍有待解决，如是否有在突触后受体的密度和谷氨酸受体亚型在突触后进补和阶段性的终端目标的差异，在此准备，但一个更好地了解这些在解剖和生理的根本区别两个电机的单位，将有助于建立更深厚的知识基础。希望是突触准备在此学到的基本原则将适用于其他各项准备工作突触，并会加强在这个小龙虾的突触模型的未来调查。 2）方法<ol><li>所有的实验都进行中型龙虾（ 克氏螯虾 ）的第一或第二的步行腿。动物被单独安置在与oxygenized水的塑料容器。动物房温度范围在13 ° C - 16 ° C。动物们喂干鱼食和每周一次的基础上改变了水。 <img src="/files/ftp_upload/2319/2319fig1.jpg" alt="图1" /> 图1：一个小龙虾步行腿和6个远段的原理图。 </li><li>在小龙虾腿走路的远端方面在解剖学上分为6段（图1）。位于小腿伸meropodite，将被隔离的神经束，接近meropodite ischiopodite联合。补药或阶段性的轴突可以Ë选择性地刺激需要后，他们所承受的生理目的。 </li><li> Cooper和Cooper（2009）描述的初始剥离的一些方面，包括揭露meropodite地区内的揭幕战中运动神经元励磁的方法，但他们的描述不提供保护免受损害伸肌所需的照料，因为它是没有需要解决的开门红肌肉准备。为了保护伸，第一或第二的步行腿的小龙虾从，测量体长6-10厘米（阿查法拉亚生物供应公司，Raceland，LA），通过诱导动物肢体有力捏automize远端ischiopodite段骨折平面。腿放在夹层板与侧（外侧）面对观众。腿转身，直到观众可以肯定外（外侧）朝上夹层板，通常与拱形侧（图2）。配售一块纸巾的腿，使得更容易打开的准备，而这些削减。 <img src="/files/ftp_upload/2319/2319fig2.jpg" alt="图2" /> 图2：侧方的meropodite，通常是拱形侧，是夹层板朝上.. </li><li>用手术刀刀片断路器和持有人，一个锋利的刀片是用来蚀刻的角质层，直到刚刚切割meropodite段在图3所示的模式通过。护理是采取不削减太远meropodite carpopodite联合远端背侧腹侧切开。 <img src="/files/ftp_upload/2319/2319fig3.jpg" alt="图3" /> 图3：行作为建议图案蚀刻角质层窗口meropodite段。 放置在盐水的准备工作。解剖盘应该有一个底部Sylgard（道康宁）涂层（厚1cm）。Sylgard是用于使昆虫引脚可分为卡仍然持有的准备。在这一点上，脚被卡在中间的carpopodite部分，并在背方面ischiopodite段（图4）。解剖的准备沐浴在标准的小龙虾生理盐水，范Harreveld的解决方案（1936年），这是205氯化钠修改; 5.3KCl; 13.5 氯化钙2 · 2H 2 O; 2.45 氯化镁2 · 6H 2 O 5 HEPES和调整，以pH值7.4（毫米）。 <img src="/files/ftp_upload/2319/2319fig4.jpg" alt="图4" /> 图4：削减窗口meropodite段升空。注意解剖引脚的位置。 </li><li>在远端地区的角质层轻轻地抬起和肌纤维被切断从角质层，对基地的腿招。角质层可以升空。 </li><li>在meropodite carpopodite联合apodeme（肌腱）被切断。一针放在屈肌腱的内表面切（图5）。这是削减用镊子退出解除在尾方向（图6），暴露的主要腿部神经和伸肌和屈肌肌腱，然后捏。 <img src="/files/ftp_upload/2319/2319fig5.jpg" alt="图5" /> 图5：屈肌apodeme取代用大头针屈突出。 <img src="/files/ftp_upload/2319/2319fig6.jpg" alt="图6" /> 图6：屈肌apodeme被切断，小心拆除，以免损坏主要小腿神经。 </li><li>主要的腿神经被切断meropodite carpopodite联合，并仔细地拉了过来伸肌回。内侧肌肉的表面是在本研究中使用。从主小腿神经伸肌的神经分离，可以提高轻轻拉动的主要腿部神经的远侧断端一侧的准备。剥离时伸肌主要小腿神经轴突的小树枝可能需要削减。这些都是从抑制运动神经元伸肌分支。较大的分支从附近的meropodite近端的小腿神经丛是利益的小神经束。 <img src="/files/ftp_upload/2319/2319fig7.jpg" alt="图7" /> 图7：主要的腿神经被切断，近端方向拉。 可以看出，这种神经束与亚甲基蓝染色（图8）或4 - DI - 2 - ASP荧光染色（图9）。 <img src="/files/ftp_upload/2319/2319fig8.jpg" alt="图8" /> 图8：伸肌与亚甲蓝染色。注意轴突的分支和两个随时可见的神经轴突内。红色箭头丹麦的神经轨道。 <img src="/files/ftp_upload/2319/2319fig9.jpg" alt="图9" /> 图9：4 - DI - 2 - ASP的运动神经染色轴突。进补轴突是由于线粒体含量增加更明亮可见。 <img src="/files/ftp_upload/2319/2319fig10a.jpg" alt="图10A" /> 图10A：4 - DI - 2 - ASP的染色的阶段性和补品神经元的个人终端。请注意进补终端和薄性质的阶段性终端膨体。 <img src="/files/ftp_upload/2319/2319fig10b.jpg" alt="图10B" /> 图10B构造说<img src="/files/ftp_upload/2319/2319fig10c.jpg" alt="图10c" /> 图10C -阶段性指出</li></ol> 3）生理资料<ol><li>要观察的补药或阶段性的神经元的兴奋性突触后电位（EPSPS），孤立的轴突在神经束之一是刺激吸草刺激电极连接（图11），而在肌肉细胞内的潜力监测（斯通等 ，2008）。在70 Hz的刺激，以促进为低输出NMJs促进救灾，适用于补虚轴突或单脉冲（1赫兹）是适用于阶段性的轴突，以获得高输出大EPSPS NMJs如图所示在图12。 EPSPS都记录到计算机通过PowerLab/4s接口。 <img src="/files/ftp_upload/2319/2319fig11.jpg" alt="图11" /> 图11：刺激电极放置在一个单一的神经束内的轴突。注意绿线概述2主轴突。 <img src="/files/ftp_upload/2319/2319fig12.jpg" alt="图12" /> 图12：补药或阶段性的神经元细胞内的录音获得的突触后电位（EPSPS ）。 </li><li>在促进突触和突触抑郁性质的调查，可以接触各种实验范式与低，产量高NMJs。便利的低输出NMJs取决于频率，为约20刺激（图13）20，40和60赫兹脉冲所示。 <img src="/files/ftp_upload/2319/2319fig13.jpg" alt="图13" /> 图13：在响应了三种不同的频率20，40和60赫兹在正常的小龙虾生理盐水刺激脉冲的培养EPSPS。 </li><li>突触抑郁率的刺激频率也与高输出NMJs。图14显示了连续5赫兹的刺激郁闷NMJ超过30分钟。随着刺激频率较高，准备将抑制更为迅速（Bradacs 等 ，1997年）。 <img src="/files/ftp_upload/2319/2319fig14.jpg" alt="图14" /> 图14：EPSP的幅度在5 Hz的刺激诱 ​​发抑郁症的阶段性反应。 </li></ol> 4）量子反应<ol><li>一个开门红肌肉小龙虾（Cooper和Cooper，2009）中描述的类似的程序是用于在此准备。直接通过识别区域的神经末梢量子EPSPS记录放在宏观补丁记录电极对突触膨体流明，具有重要的染料4 -二- 2 - ASP（5微米，5分钟的治疗 ，Cooper等可视化，1995; Magrassi 等 ，1987）。可以记录沿神经末梢自发以及诱发量子反应。诱发和自发突触电位记录与宏观补丁电极（Dudel，1981;武伊托维奇等 ，1991;。Mallart，1993年）。 Kimax玻璃（外径：1.5毫米），被拉到和火抛光生产内径从10到20微米（图15）的补丁提示。 <img src="/files/ftp_upload/2319/2319fig15.jpg" alt="图15" /> 图15：宏观补丁的记录电极流明.. 电极内腔充满了沐浴液。该放大器是上述细胞内的录音中使用的相同。通过测试电流脉冲通过电极，电极和密封性能，可确定。在我们的实验中，密封电阻范围从0.3至1.0毫欧和电极电阻从0.5至1.0毫欧不等。密封电阻可以监视整个录音。 </li><li>量子事件的直接计数可能与低刺激的频率。量子事件对于每个诱发反应，可以很容易地确定为低输出端子（FigurE 16）。这些直接计数，可以帮助估计平均量子含量（德尔卡斯蒂略卡茨，1954年，Cooper 等人，1995年） 。由于诱发的高输出NMJs产生多量子诱发事件，平均振幅或地区的挠度，平均峰值振幅或面积自燃事件，可用于近似的平均量子含量 （Cooper等人。 1995年）。 <img src="/files/ftp_upload/2319/2319fig16.jpg" alt="图16" /> 图16焦距的痕迹，记录从一个阶段性和一剂强心剂NMJ 。 <img src="/files/ftp_upload/2319/2319fig17.jpg" alt="图17" /> 图17 </li></ol>

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Biol. 43, 229-246 (1965).</li><li>King, M. J. R., Atwood, H. L., Govind, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+features+of+crayfish+phasic+and+tonic+neuromuscular+junctions.">Structural features of crayfish phasic and tonic neuromuscular junctions.</a> J. Comp. Neurol. 372, 618-626 (1996).</li><li>LaFramboise, W., Griffis, B., Bonner, P., Warren, W., Scalise, D., Guthrie, R. D., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+type+specific+myosin+isoforms+in+crustacean+muscles.">Muscle type specific myosin isoforms in crustacean muscles.</a> J. Exp. Zool. 286, 36-48 (2000).</li><li>Logsdon, S., Johnstone, A. F. M., Viele, K., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+synaptic+vesicles+pools+within+motor+nerve+terminals+during+short-term+facilitation+and+neuromodulation.">Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.</a> J. Applied Physiol. 100, 662-671 (2006).</li><li>Lucas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+complex+excitable+tissues+by+their+response+to+electric+currents+of+short+duration.">The analysis of complex excitable tissues by their response to electric currents of short duration.</a> J. Physiol. 35, 310-331 (1907).</li><li>Lucas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+summation+of+propagated+disturbances+in+the+claw+of+Astacus+and+on+the+double+neuromuscular+system+of+the+abductor.">On summation of propagated disturbances in the claw of Astacus and on the double neuromuscular system of the abductor.</a> J. Physiol. 51, 1-35 (1917).</li><li>Magrassi, L., Purves, D., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+that+stain+living+nerve+terminals.">Fluorescent probes that stain living nerve terminals.</a> J. Neurosci. 7, 1207-1214 (1987).</li><li>Millar, A. G., Bradacs, H., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+relationship+between+release+probability+and+readily+releasable+vesicles+in+depressing+and+facilitating+synapses.">Inverse relationship between release probability and readily releasable vesicles in depressing and facilitating synapses.</a> J. Neurosci. 22, 9661-9667 (2002).</li><li>Millar, A. G., Zucker, R. S., Ellis-Davies, C. G., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensitivity+of+neurotransmitter+release+differs+at+phasic+and+tonic+synapses.">Calcium sensitivity of neurotransmitter release differs at phasic and tonic synapses.</a> J. Neurosci. 25, 3113-3125 (2005).</li><li>Msghina, M., Govind, C. K., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+structure+and+transmitter+release+in+crustacean+phasic+and+tonic+motor+neurons.">Synaptic structure and transmitter release in crustacean phasic and tonic motor neurons.</a> J. Neurosci. 18, 1374-1382 (1998).</li><li>Msghina, M., Millar, A. G., Charlton, M. P., Govind, C. K., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+entry+related+to+active+zones+and+differences+in+transmitter+release+at+phasic+and+tonic+synapses.">Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses.</a> J. Neurosci. 19, 8419-8434 (1999).</li><li>Mykles, D. L., Medler, S. A., Koenders, A., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillar+protein+isoform+expression+is+correlated+with+synaptic+efficacy+in+slow+fibres+of+the+claw+and+leg+opener+muscles+of+crayfish+and.">Myofibrillar protein isoform expression is correlated with synaptic efficacy in slow fibres of the claw and leg opener muscles of crayfish and.</a> 205 (4), 513-522 (2002).</li><li>Sparks, G., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT+offsets+homeostasis+of+synaptic+transmission+during+short-term+facilitation.">5-HT offsets homeostasis of synaptic transmission during short-term facilitation.</a> J. Applied Physiol. 96, 1681-1690 (2004).</li><li>Sohn, J., Mykles, D. L., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomical,+physiological+and+biochemical+characterization+of+muscles+associated+with+the+articulating+membrane+in+the+dorsal+surface+of+the+crayfish+abdomen.">The anatomical, physiological and biochemical characterization of muscles associated with the articulating membrane in the dorsal surface of the crayfish abdomen.</a> J. Exp. Zool. 287, 353-377 (2004).</li><li>Tsien, R. W., Harata, N. C., Choi, S. W. P. y. l. e., Aravanis, A. M., AM, G. C. h. e. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicles,+quanta+and+synaptic+plasticity.+Molecular+Mechanisms+of+synaptic+function.">Vesicles, quanta and synaptic plasticity. Molecular Mechanisms of synaptic function.</a> , (2001).</li><li>Van Harreveld, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiological+solution+for+freshwater+crustaceans.">A physiological solution for freshwater crustaceans.</a> Proc. Soc. Exp. Biol. Med. 34, 428-432 (1936).</li><li>Van Harreveld, A., Wiersma, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triple+innervation+of+the+crayfish+muscle.">The triple innervation of the crayfish muscle.</a> Proc. Natl. Acad. Sci. USA. 22, 667-667 (1936).</li><li>Wiersma, C. A. G., Van Harreveld, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+the+frequency+of+stimulation+on+the+slow+and+fast+contraction+in+crustacean+muscle.">The influence of the frequency of stimulation on the slow and fast contraction in crustacean muscle.</a> Physiol. Zool. 11, 75-81 (1938).</li><li>Wojtowicz, J. M., Smith, B. R., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+recruitment+of+silent+synapses.">Activity-dependent recruitment of silent synapses.</a> Ann. N.Y. Acad. Sci. 627, 169-179 (1991).</li></ol>

我们已经证明，在这份报告中，如何剖析，记录和量化在一个独特的小龙虾神经肌肉的准备，在高和低输出端子支配的肌纤维突触反应。在小龙虾的神经肌肉筹备工作提供了超过脊椎动物神经肌肉交界处的许多优点，因为只有少数的兴奋性运动神经元支配的肌肉，而且因为神经元是从准备到识别的准备（阿特伍德，1976）。此外，兴奋性神经递质谷氨酸，和分级的兴奋性突触后电位（EPSP），因此，分级事件的生物物理特性类似于脊椎动物的中枢神经系统内神经元的树突。然而，量子电流，可以直接监测在突触后的网站（Cooper 等人 ，1995年）。 重复5赫兹的阶段性神经刺激产生几分钟后，成为大大郁闷的大EPSPS。这种类型的抑郁症是常见于节肢动物阶段性的神经肌肉接头（阿特伍德和库珀，1996）。旁边的阶段性终端的补药终端的存在，允许一个突触后的目标是评估是否大大阶段性的运动神经元的抑郁症，期间和之后的修改。此外，低输出端子提供了一个很好的准备，调查机制的基础突触便利（2008年德赛- Shah 等人，;。德赛Shah和库珀，2009 ） 高输出端子提供了一个发挥地面调查突触抑郁症和恢复过程的速度调制。访问和活菌制剂，应帮助破译突触抑郁症背后的机制。在这个小龙虾腿伸准备，外源性羟色胺的应用，是另一种工具来调查突触抑郁症和潜在的进一步调查动员进入突触小泡池的手段恢复。这种准备提供了几个实验的优势，因为个人的阶段性和补品的运动神经元支配的肌纤维。 由于血清素增加，诱发刺激释放的囊泡数量，因为它促进在抑郁症的恢复，很明显，有一些小泡池，为加强与融合的可能性-羟色胺目前的调制（约翰斯顿等人，2008年劳格斯登等，2006;火花等，2004）。低频刺激，而不是一个低迷的状态，这可以解释在突触前神经末梢的囊泡池的动态模型还需要考虑这种准备可能的实验之间的各种协议。 人们已经注意到，在其他系统中，约30％的小泡池，经历了一个快速的回收利用，而其余的回收囊泡通过内质网（Harata等，2001;。钱学森等人通过传统的缓慢回收路径。 ，2001）。这种双重路径，也可以在这个系统中存在。如果ATP是缺乏在突触前终端的低迷状态，然后对接和脱开可能无法出现，因此，许多卸载囊泡停留在表面的突触。由于越来越多的小泡是迅速释放终端接触到5羟色胺，它是可行的，更多的是易释放池（RRV）内所载。然而，在低迷的状态，与减少ATP的对接和脱开可能受阻，甚至在5羟色胺，再次离开卸载囊泡在突触表面的存在。由于初步数据表明，随着时间的推移释放更多的囊泡延长5羟色胺的暴露和刺激，小泡池分布的快与慢的回收路径可能是倾斜的主管重新释放囊泡 （斯通等。 ，2008;评论德赛- Shah 等人，2008;德赛Shah和库珀，2009） 随着性突触后电流和措施，从运动神经终端的定义的地区单量子联络macropatch录音技术，可以提问，以确定是否突触抑郁症是作为一个被释放的囊泡的结果，或者因为改动的功能发生突触后受体。它已被证明的囊泡平等钙曝光（Miller 等人在这个准备的阶段性NMJ融合更为敏感 。2005年），这可能占较高意味着量子阶段性终端（Msghina等内容 。 1998年，1999年）。此外，中的钙结合蛋白frequenin的差异（Jeromin等 ，1999）和超微结构（King等，1996）作出贡献的差突触效能（库珀等，2003）。 一些以前的研究探索的伸肌的肌肉纤维表型（Bradacs等人 ，1997 年 ，Cooper 等人 ，2003 年 ） 。肌肉分化调节混合纤维类型的小龙虾，纯粹是补药和阶段性的光纤类型，比较喜欢钝地支配小腿伸，可以提供线索，肌表型的表达和调控（LaFramboise等，2000 ;孙某等。 人，2000年Griffis 等，2001; Mykles 等，2002）。 许多根本性的问题有待解决在神经生物学，该制剂可能有助于解决其中一些。在该领域的兴趣今天的一个，可能是的腿伸上前几个主题包括：1）确定的细胞机制，基础在高输出终端突触抑郁症（抑郁由于减少钙+入门，缺乏主管易释放囊泡（RRV）池，和/或改变突触后的接受吗？）2）确定的5 - 羟色胺的机械作用后，诱导突触抑郁症的应用，以促进更快的复苏，以及3）确定是否量子形状从刺激阶段性终端产生的电流，正在改变在抑郁症的感应，解决突触抑郁前和突触后成分。 这NMJ正在进行的研究和未来的调查是非常重要的，因为它使我们能够获得相关信息，解决机制，在释放地点直接测量突触的性能下属。目前这方面的研究提供5 - 羟色胺和小泡池的动态调制突触抑郁症有关的信息。等议题涉及到有关的所有神经系统的突触传递的根本基础。

<ol><li>小龙虾（ 克氏螯虾 ）。阿查法拉亚生物供应公司，Raceland，洛杉矶，美国。 </li><li>标准小龙虾生理盐水： <ul><li>修改从范Harreveld的解决方案（1936年）。 （205毫米）氯化钠氯化钾5.3; 13.5氯化钙2 · 2H 2 O; 2.45氯化镁2 · 6H 2 O 5 HEPES和调整pH值7.4 </li></ul></li><li>解剖工具：精细＃5镊子，精剪，刀刀座，昆虫引脚＃26002-20（所有获得精细科学的工具（美国）公司，373 - G的复古公园径，福斯特市，CA 94404-1139） </li><li> Sylgard涂层夹层盘，录音菜吸电极构造或使用吸电极和花药操盘举行吸电极。 </li><li>解剖显微镜与细胞内的录音变焦功能。对于一个正直的目标（4倍和20倍）的复合式显微镜是使用可视终端的联络记录。人们需要一种汞光源。 </li><li>标准细胞内的放大器和A / D板线录制到计算机。电信号记录上线到PowerLab/4s接口（澳大利亚ADInstruments，）。我们使用标准软件名为图表或范围ADInstruments。 </li><li>化学品： <ul><li>我们用一个重要的荧光染料，4 [4（二乙氨基）苯乙烯基] N methylpyridinium碘（4 DI 2门冬;分子探针，尤金，OR），可视化膨体（Marigassi等，1987; Cooper 等人，1995 年 。 ）。从Sigma公司（圣路易斯，密苏里州）获得所有生理盐水化学品。 </li></ul></li><li>细胞内的录音，我们使用玻璃毛细管（从金控，不伦瑞克，ME，04011，美国的目录＃30-31-0）和聚焦macropatch电极，我们使用Kimax - 51，金布尔产品艺术。 34502号，内径0.81.1毫米，长度100mm。细胞内的电极应该有一个20至30毫欧电阻。 macropatch电极构造掰后的精尖是从一个电极拉马的玻璃的一角。折断提示需要一个直径约20μM清洁垂直打破。笔尖再热抛光，内径10μm左右。电极轴，然后跑过来一个加热元件，以使其一个渐进的弯曲约45度弯曲。这将产生一个以上的神经末端的微型机械臂的角度平面或垂直的电极流明，会产生约45度的准备。 </li></ol>

physiological recordings of high and low output nmjs on the crayfish leg extensor muscle

我们详细解释如何进行揭露和高（相位）和低（补品）输出运动神经元支配伸肌在一个小龙虾腿走路的突触反应的电生理记录。在目前的阶段性和补品神经末梢的生理和形态明显的差异。进补轴突包含有更多的线粒体，使其能够采取一个重要的污点更为激烈的阶段性轴突。进补终端有静脉曲张，和阶段性的终端是丝状。进补终端突触效能低，但戏剧性的便利反应。相反，阶段性的终端在量子疗效高，但显示高频刺激突触抑郁。量子输出是衡量一个焦点macropatch电极直接放置在可视化的神经末梢。无论是阶段性和补品终端支配同一肌肉纤维，这表明，在神经元固有的分歧，而不是差逆行反馈的肌肉，帐户的形态和生理分化。

Watch this Scientific Journal Video about Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle at JoVE.com

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

本文演示了如何进行突触反应的电生理记录的伸肌在一个小龙虾腿走路和神经末梢如何可视化显示的高和低输出神经末梢总值的形态差异。

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 ...

physiological-recordings-high-low-output-nmjs-on-crayfish-leg

Research

JoVE Journal

Neuroscience

11.6K 次观看.  University of Kentucky. 本文演示了如何进行突触反应的电生理记录的伸肌在一个小龙虾腿走路和神经末梢如何可视化显示的高和低输出神经末梢总值的形态差异。

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

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的：一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

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 ...

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

高密度的自由移动，使用基于聚酰亚胺的微电极的小鼠的脑电图记录

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

小龙虾的游泳足系统：实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...