我们感谢乔斯Bu​​rgert帮助有一些数字。我们感谢英戈SELBACH（和组"Edelkrebsprojekt北威州"），为他的努力提供实验室与实验动物。我们感谢安娜·C·施奈德校对书稿的第一个版本。这项研究是由埃米诺特DFG授予SM 206 / 3-1和科隆大学女教师启动资助。

这解剖过程是按照22欧洲共同体理事会指令次 2010年9月（2010年/ 63 / EU）。 1.准备<ol><li>小龙虾获得男女≥8厘米大小的， 螯leniusculus（德纳）。确保动物是至关重要的，腹部和四肢腹部都完好无损。 </li><li>小心检查甲壳而这个角质层又粗又硬。前和postmolt动物具有柔软甲壳和不适合的实验，因为在蜕皮过程中的许多参数改变（ 例如，减少在运动活性）。 </li><li>组装解剖过程中使用的所有工具和材料，钉扎和神经索的desheathing 图2中示出并在所提供的补充剂列出。 </li></ol><img alt="图2" src="/files/ftp_upload/52109/52109fig2highres.jpg" /> 图2: <s仲&gt;材料和工具用于清扫，牵制和神经索的desheathing。 （1）大水桶装满冰块; （2）小龙虾盐水; （3）盐水分配器; （4）解剖显微镜; （5）夹层菜; （6）强大的剪刀; （7）镊子（8）弹簧剪刀; （9）培养皿内衬明确SYLGARD; （10）固定销; （11）冷灯源。 2.总解剖<ol><li>麻醉动物在冰上15 - 20分钟。进行总夹层的第一部分在水槽附近的实验室工作台上，因为它包括一个放血步骤和试样需要定期用小龙虾盐水在手术过程中漂洗。 </li><li>保持动物腹侧和使用强大的剪刀在靠近胸部（ 图3-1）他们的基地削减双方的爪子。拆下左，右尾肢（ 图3-2）。 </li><li>将动物腹侧的解剖盘内衬黑色SYLGARD。的Eleva德头胸部通过在尾节（ 图4A）下插入冰和引脚腹部。 </li><li>填充生理盐水饮水机用〜60毫升冷冻小龙虾生理盐水。灌注小龙虾通过爪孔（ 图4B）冷盐水。多余的盐水将通过削减在腹足流掉。覆盖小龙虾与放血过程中的冰</li><li>斩首动物用单个横向切割只是后侧到使用强剪刀（ 图5A）的动物的眼睛。删除所有腿走路附近的基地关节， 如图5B所示。 </li><li>隔离与来自头胸部的其余部分的最后胸段腹部。通过插入剪刀的尖端插入所述第二行走腿的开口和切削到相对侧使在第二行走腿的水平（胸段3）的第一切口。 （ 图6A-1）。 </li><li>扩展这个首次降息双方通过吨他头胸部（ 图6A-2）。 </li><li>翻转动物开放做出一些内脏可见。推突出消化腺（ 图6B-3）在试样的前部，并使用镊子从腹腔取出生殖器官。 </li><li>除去头胸部（ 图6C）的前部。用横向切割以除去甲壳的横向部件，覆盖鳃，对剩余的胸部（ 图6D-4）的两侧。取出鳃和冲洗用冷盐水标本。 </li><li>通过胸骨板的整个长度继续进行切割夹层， 如图6E-5表示。使这个在削减和pleuron游泳足（ 图6E红色标记）之间的最大横向位置。继续与同一切对方。冲洗用冷盐水的样品。 </li><li>根据dissectio进行清扫剩余ñ显微镜。放置小龙虾的腹部腹侧在解剖盘内衬黑色SYLGARD和填充小龙虾生理盐水，使其覆盖所述样品。 注:以下步骤（2.12-4.8）包含适用于右手实验者的方向指示。在下面的步骤（2.12-6.8）它用冷盐水代替小龙虾盐水在规则的间隔，每次20-30分钟，以保持对神经系统的健康是非常重要的。 </li><li>修复与昆虫针试样后方的尾节，并在前方的背甲的遗体。放置试样，以使尾节点的左侧和平行于该表的边缘。 </li><li>使用左手粗钳抓住通过行走的腿开口（ 图7A）和拉标本开（ 图7B白色箭头）。识别大型两背侧屈肌股线（ 图7B-1和C-1），并切断其腹如图7B基础。 </li><li>识别胸骨动脉（ 图7C-2），一个从背侧（心脏），以腹侧，在第 4胸椎节段。该动脉位于右侧的神经索（ 图7C-3）上面，它的神经线下的项目之前，形成腹动脉。 </li><li>横切胸骨动脉。这表现在图7C中 ，首先解除动脉，用剪刀的一个叶片，并且仅切割时的腹侧位于神经索是可见的。 </li><li>前方固定背屈肌（向右侧）。这应该在最大伸展位置来完成与引脚，因此他们不会挡住视野和标本仍然捉襟见肘。当背肌肉股被固定（ 图8-1）中，第一腹神经节，A1和A2，与相 ​​关的神经N1，N2和N3是可见的（ 图8）。 </li><li>使用镊子在左手抓住规范imen在一个开口步行腿。纵观以下步骤剥离轻轻拉，以保持标本打开。 </li><li>横切的神经N3从神经线最远的位置。 （ 图8-3）。 </li><li>切开屈肌肌肉接近腹表皮内突， 如图8-4所示 。请注意不要损伤神经线或神经N2。 </li><li>重复步骤2.18和2.19的神经N3，其余腹腔神经节A2到A5的屈肌肌肉。 </li><li>在最后的腹神经节，A6，切背屈肌，从腹侧表皮内突和试样应在图9的样子，如图所示。 </li><li>切开胸骨板后，以A6（ 图9-1）的神经，保持腹部的一部分（ 图9-2）。丢弃背侧部与屈肌肌肉（ 图9-3）。通过行走的腿的开口与销前方固定胸骨板，并且向后到A6。 </li></ol> 3.精细解剖<ol><li>将样品与前部引导离开并朝向表&#39;边缘的后部，在显微镜下。 </li><li>使用镊子， 如图10A除去cephalothoracic胸骨的最前部分。 注:胸神经节和相关的神经得到部分弥补了腿部肌肉和cephalothoracic燕。所述cephalothoracic腹板形成骨架分隔行走腿的侧向位于空腔彼此并从中间腔，其中腹神经索驻留。 </li><li>切， 如图10B所示的剩余外骨骼结构-1和B2之间的肌肉。使用镊子抓住并提起腹神经索（ 图10C）的前端。 注:神经线将在此过程中被损坏，以便避免拿起NERVË线多次。 </li><li>切胸神经横向同时抬起神经索（图10C-3）。保持这些神经在适当的长度牵制。除去神经节，将其拾起用钳子的链挤压部，通过切除所有的组织前到T4（ 图10C-4）。 </li><li>将样品与前部的左侧和集中在A1中。切A1的神经N1和N2在合适的长度（最大1厘米）对于钉扎出来。 </li><li>着眼于A2和识别神经N1，N2和该段的N3（ 图11）。腹神经节A2-A5的神经N1驻留2胸骨表皮infoldings之间在各段（ 图11A-1）和被覆盖的肌肉。做一个切沿胸骨后表皮内折。开始在腹部的侧缘，并继续朝向中线， 如图11A。 </li><li>如果目标N1仍然是海湾红色与组织， 如图11B（红色箭头），切断跨肌束，但此时前两者胸骨表皮infoldings和神经N1（ 图11B-2）。 </li><li>切断神经N1作为远侧越好（ 图11C-3）。神经N1为完全可见，前部和后部支可识别（ 图11C）。 </li><li>继续向健侧神经N1和第一切开肌肉沿胸骨后表皮内折，从内侧，靠近神经节（ 图11D）。如果神经仍然覆盖由组织，切横跨肌束，但此时前两者胸骨表皮infoldings和神经N1，类似于图11B-2。切断神经N1作为远端越好。 </li><li>切断该神经节的神经N2到合适的长度（约0.5厘米）对钉扎。 </li><li>重复步骤3.7-3.11为A3-A5的神经。 </li><li>切仪神经节A6的VES的远侧尽可能（ 图12A）。使用镊子抢A6多个神经，解除这一节，并开始从胸骨板隔离神经节链。 </li><li>同时抬起神经线，这表现在图12B（白色箭头），轻轻在前面的方向拉。作为个别神经节被提升，取出腹侧动脉可附着到神经索（ 图12C）的腹侧。继续这个（轻轻）拉切序列，直到神经索是完全隔离的。 </li><li>神经节的分离链转移至培养皿内衬清晰SYLGARD和填充小龙虾的盐水（ 图12D）。 </li></ol> 4.管脚的神经索到培养皿注:使用小针切不锈钢线材（见补充）引脚神经线。触摸只能用产钳结束神经，不SQUEEZe本连接词或神经节。 <ol><li>针神经节在一条直线上的链条，同时施加轻柔舒展。 </li><li>安排在培养皿背侧的神经索朝上（ 图13中黑线）。神经节的腹侧可以标识通过其凸;背侧是平坦的。针胸神经向两侧。继续与A6的神经，沿其纵向轴线伸展的神经索。 </li><li>引脚输出A1的神经，在90°角相对于神经索一个。 </li><li>着手A2和引脚神经N2在35-45°相对于神经索（ 图1A）的角度。 </li><li>钉扎这表现在图14中之前分离的神经N1在其前部和后部的分支，使用两对细镊子的拾起带有一对钳子的前部，并与其他的神经N1的后支。小心挑选只有最前端S中的神经分支。现在拉他们仔细分开。 </li><li>针神经N1的前支在90°角相对于神经索（ 图1A）一个。脚前N1分支和神经之间的N2 N1神经的后支。 </li><li>重复步骤4.4-4.6为神经节A3-A5的神经。而行了吧神经索牵拉它在纵向和横向方向。 </li></ol> 5. Desheathing神经节<ol><li>将制剂以这样的方式即实验者的手总是搁在一个稳定的平面，以避免振荡。为了desheath神经节从下面照亮的神经线。 </li><li>专注于任何腹神经节A1到A5。用细弹簧剪刀，使通过节套一小横向切割，后到神经节的神经N2和N3（ 图15A红色箭头）。 </li><li>使用非常˚F拿起节护套INE镊子和横向切割穿过上述联结套管，如在图15A-1表示。小心不要挤压或剪切的神经索用剪刀。 </li><li>仍持有和解除神经节护套用钳子继续削减它沿神经节（ 图15B-2，-3）的横向边界。移除护套。可替代地将其固定到联结的两侧，使得它被固定，但在神经索未挤压的方法。 </li><li>所有腹节A1至A5重复步骤5.2-5.4。 </li><li> Desheathe胸神经节和以类似的方式在神经节的A6。为了desheathe A6开始前的神经节，并继续在后方向。针A6的神经节护套神经节的链的后端。 </li></ol> 6.外记录，从运动神经元<ol><li>组装用于显示我外记录所有的工具和材料Q 图16B和上市的补充。在记录设置的概况示于图16A。启动在该实验中（ 图16C）使用的所有电子设备，从而使放大器可以预热至少30分钟记录之前。打开电脑并启动录音软件。 </li><li>放置神经节上的显微镜表的链和从下方照亮。插入记录电极插入SYLGARD接近目标神经，并在附近的位置上的参考电极，但横向于神经节（ 图17A和B）。周围弯曲的记录电极（ 图17C）的目标神经。 </li><li>伸展的神经咯，以确保电极与神经之间的接触，并将其管脚到侧（F igure 17D）。固定用橡皮泥电极电缆连接到显微镜表，所以它们留在所需的位置。 </li><li>使用注射器填充有凡士林和一个20号针头（具有圆形的尖端）（ 图17E-3）以分离从浴液目标神经。第一dAb一些凡士林在记录电极周围的SY​​LGARD。其结果是一层凡士林覆盖在记录电极（ 图17E-4）的附近的SYLGARD。小心不要直接轻拍到神经和避免气泡在这一层。 </li><li>密封用凡士林来自各方面的记录电极至盐水（ 图17F）的表面水平。 </li><li>重复此过程为所有目标神经的活动进行监测。 </li><li>开始录制。使用连续或间隙自由获取模式和为5 kHz的取样速率。设置外放大器下列参数;争取到1000（放大信号1000次，照顾到包括在收购软件设置这个参数放大）的300赫兹（低胸）到2000赫兹（高切）达带通滤波器的范围。 </li></ol>

<ol>
	<li>Hughes, G. M., 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+Co-Ordination+of+Swimmeret+Movements+in+the+Crayfish,+Procambarus-Clarkii+(Girard).">The Co-Ordination of Swimmeret Movements in the Crayfish, Procambarus-Clarkii (Girard).</a> J Exp Biol. 37 (4), 657-670 (1960).</li><li>Mulloney, B., Smarandache, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifty+Years+of+CPGs:+Two+Neuroethological+Papers+that+Shaped+the+Course+of+Neuroscience.">Fifty Years of CPGs: Two Neuroethological Papers that Shaped the Course of Neuroscience.</a> Front Behav Neurosci. 4, 45 (2010).</li><li>Murchison, D., Chrachri, A., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Separate+Local+Pattern-Generating+Circuit+Controls+the+Movements+of+Each+Swimmeret+in+Crayfish.">A Separate Local Pattern-Generating Circuit Controls the Movements of Each Swimmeret in Crayfish.</a> J Neurophys. 70 (6), 2620-2631 (1993).</li><li>Mulloney, B., Hall, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+crayfish+abdominal+ganglia.+III.+Swimmeret+motor+neurons.">Functional organization of crayfish abdominal ganglia. III. Swimmeret motor neurons.</a> J Comp Neurol. 419 (2), 233-243 (2000).</li><li>Davis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lobster+Righting+Responses+and+Their+Neural+Control.">Lobster Righting Responses and Their Neural Control.</a> Proc R Soc Ser B-Bio. 170 (1021), 435-456 (1968).</li><li>Mulloney, B., Smarandache-Wellmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+the+crustacean+swimmeret+system.">Neurobiology of the crustacean swimmeret system.</a> Prog Neurobiol. 96 (2), 242-267 (2012).</li><li>Huxley, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The crayfish: An introduction to the study of zoology. , (1980).</li><li>Smarandache-Wellmann, C., Weller, C., Wright, T. M., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+types+of+nonspiking+interneurons+in+local+pattern-generating+circuits+of+the+crayfish+swimmeret+system.">Five types of nonspiking interneurons in local pattern-generating circuits of the crayfish swimmeret system.</a> J Neurophys. 110 (2), 344-357 (2013).</li><li>Skinner, F. K., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersegmental+coordination+of+limb+movements+during+locomotion:+mathematical+models+predict+circuits+that+drive+swimmeret+beating.">Intersegmental coordination of limb movements during locomotion: mathematical models predict circuits that drive swimmeret beating.</a> J Neurosci. 18 (10), 3831-3842 (1998).</li><li>Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=During+fictive+locomotion,+graded+synaptic+currents+drive+bursts+of+impulses+in+swimmeret+motor+neurons.">During fictive locomotion, graded synaptic currents drive bursts of impulses in swimmeret motor neurons.</a> J Neurosci. 23 (13), 5953-5962 (2003).</li><li>Smarandache-Wellmann, C., Gr&#228;tsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+coordination+in+distributed+neural+circuits:+Encoding+coordinating+information.">Mechanisms of coordination in distributed neural circuits: Encoding coordinating information.</a> J Neurosci. 34 (16), 5627-5639 (2014).</li><li>Mulloney, B., Hall, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+commissural+interneurons+integrate+information+from+intersegmental+coordinating+interneurons.">Local commissural interneurons integrate information from intersegmental coordinating interneurons.</a> J Comp Neurol. 466 (3), 366-376 (2003).</li><li>Mulloney, B., Harness, P. I., Hall, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursts+of+information:+Coordinating+interneurons+encode+multiple+parameters+of+a+periodic+motor+pattern.">Bursts of information: Coordinating interneurons encode multiple parameters of a periodic motor pattern.</a> J Neurophys. 95 (2), 850-861 (2006).</li><li>Smarandache, C., Hall, W. M., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+Rhythmic+Motor+Activity+by+Gradients+of+Synaptic+Strength+in+a+Neural+Circuit+That+Couples+Modular+Neural+Oscillators.">Coordination of Rhythmic Motor Activity by Gradients of Synaptic Strength in a Neural Circuit That Couples Modular Neural Oscillators.</a> J Neurosci. 29 (29), 9351-9360 (2009).</li><li>Smarandache-Wellmann, C., Weller, C., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Coordination+in+Distributed+Neural+Circuits:+Decoding+and+Integration+of+Coordinating+Information.">Mechanisms of Coordination in Distributed Neural Circuits: Decoding and Integration of Coordinating Information.</a> J Neurosci. 34 (3), 793-803 (2014).</li><li>Chrachri, A., Neil, D., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependent+Responses+of+2+Motor+Systems+in+the+Crayfish,+Pacifastacus+leniusculus.">State-Dependent Responses of 2 Motor Systems in the Crayfish, Pacifastacus leniusculus.</a> J Comp Physiol A. 175 (3), 371-380 (1994).</li><li>Chrachri, A., Neil, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+and+Synchronization+between+2+Abdominal+Motor+Systems+in+Crayfish.">Interaction and Synchronization between 2 Abdominal Motor Systems in Crayfish.</a> J Neurophys. 69 (5), 1373-1383 (1993).</li><li>Skinner, 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+Structure+of+the+4th+Abdominal-Ganglion+of+the+Crayfish,+Procambarus-Clarki+(Girard)+II.+Synaptic+Neuropils.">The Structure of the 4th Abdominal-Ganglion of the Crayfish, Procambarus-Clarki (Girard) II. Synaptic Neuropils.</a> J Comp Neurol. 234 (2), 182-191 (1985).</li><li>Skinner, 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+Structure+of+the+4th+Abdominal-Ganglion+of+the+Crayfish,+Procambarus-Clarki+(Girard)+I.+Tracts+in+the+Ganglionic+Core.">The Structure of the 4th Abdominal-Ganglion of the Crayfish, Procambarus-Clarki (Girard) I. Tracts in the Ganglionic Core.</a> J Comp Neurol. 234 (2), 168-181 (1985).</li><li>Mulloney, B., Tschuluun, N., Hall, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectonics+of+crayfish+ganglia.">Architectonics of crayfish ganglia.</a> Microsc Res Techniq. 60 (3), 253-265 (2003).</li><li>Braun, G., Mulloney, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+modulation+of+the+swimmeret+motor+system+in+crayfish.">Cholinergic modulation of the swimmeret motor system in crayfish.</a> J Neurophys. 70 (6), 2391-2398 (1993).</li><li>Davis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motoneuron+Morphology+and+Synaptic+Contacts+-+Determination+by+Intracellular+Dye+Injection.">Motoneuron Morphology and Synaptic Contacts - Determination by Intracellular Dye Injection.</a> Science. 168 (3937), 1358-1360 (1970).</li><li>Altman, J. S., Tyrer, N. M., Strausfeld, N. J., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filling+Selected+Neurons+with+Cobalt+through+Cut+Axons.">Filling Selected Neurons with Cobalt through Cut Axons.</a> Neuroanatomical Techniques. , 373-402 (1980).</li></ol>

The authors declare that they have no competing financial interests.

<html>小龙虾的解剖和它们的腹神经节以前已经描述5，18，19，20和建议之前夹层熟悉它们，以避免的重要神经切割。 关键是要保持的制备在温度低于23℃，以防止分离的神经索的降解。这可以容易地通过更换浴液每20-30分钟用冷小龙虾盐水来实现。在这些情况下，可用于电生理实验长达12小时神经节的链。 有时候，游泳足运动神经元处于非活动状态，并显示没有自发的爆发活动。胆碱能激动剂卡巴用于诱导爆破活动在这些制剂中。溶于冷小龙虾盐水，1-3微米卡巴浴的应用是足够的，以激活游泳足节奏21。 ove_content"&gt;假想运动，并记录在该系统中四肢特别的协调提供了很多关于节律活动和协调的细胞性质的信息。在这个系统中的神经元组件的局部微电路8-10被识别并且另外的协调的网络被称为蜂窝详细11,14,15。从与细胞外记录进行的实验得出的结果，已经让预测的数学模型和机器人专家。 所有游泳足的神经元显示出横向神经纤维内树枝状分枝，并且可以从这些树突细胞内被记录下来。在图20中 ，示出了PS的运动神经元的细胞内记录。对于这种尖锐的微电极（见补充剂）填充用1M醋酸钾+ 0.1M的氯化钾（35之间电极电阻 - 45MΩ）置于上方侧向神经纤维。振动膜电位为W从PS和RS运动神经元的树突ELL尖峰可当电极插入神经N1的根部附近进行记录。 染色单个神经元（ 例如，运动神经元）在细胞内， 如图21所示 ，锋利微电极必须另外填充有荧光染料（ 例如，1％的葡聚糖德克萨斯红，见补充剂）溶解在1M醋酸钾+ 0.1M的氯化钾。到细胞内填充的运动神经元通过离子电渗，偏光1 nA的脉冲250毫秒2 Hz的持续时间被施加至少10分钟。对正电荷的染料使用去极化脉冲和用于带负电荷的染料使用超极化脉冲。再填充导致更强的标记神经元。对于可视化的神经线必须固定，脱水，并安装22。 一种方法进行染色的运动神经元的一个池是使用回填从神经N1（ 图22）4，23。放置在神经周围N1的后支凡士林的阱回填的PS的运动神经元的池。染色的RS电动机的池神经元以及被放置在神经N1的前支。在井的小龙虾盐水换成双蒸2 O.然后切这根神经井里，让它15分钟的DDH 2 O的在室温（RT）。或5％的NiCl 2 - -溶液（溶解在双蒸2 O）和关闭井凡士林此后，无论是用5％ 氯化钴取代DDH 2 O 的孔中。样品需要在4℃进行温育至少15小时。开的孔，去除染料并用盐水冲洗样品3次。沉淀出的Ni 2+或Co 2+离子与10滴Dithiooxamid在培养皿每10毫升小龙虾的盐水（溶解在100％EtOH中的饱和溶液），罩20分钟，在室温下。镍离子 -离子会沉淀蓝镍（SCH）和Co 2+ </suP&gt; - 离子橙色的CoS。神经线必须固定，脱水，并安装在顺序光学显微镜配备有相机下以获得标记的细胞的图像。

小龙虾的游泳足发球局中姿态控制功能，并有节奏地敲打当动物往前游，通风的洞穴或女性充气鸡蛋5，6，信号小龙虾， 螯leniusculus的游泳足，是成对出现的，从第二到第五腹段，用一肢上腹部7的每一侧。对中枢神经系统产生自身的节律马达图案驱动在完整的动物，以及在分离的神经索制备的游泳足运动。当没有感官反馈或降序输入本公司生产的节奏马达模式被称为假想运动1,2，在游泳足系统该电动机模式中不从在完整动物测得的游泳足的活性的任何参数不同。 每个游泳足的运动是通过将位于和限制为一个c-微电路驱动orresponding hemiganglion 1 - 3在每个微电路是有规律生成内核包括五个确定非扣球的interneurons。它们可以在功能特征为两种动力冲程抑制剂（IPS）或回击抑制剂（IRS）8。这些IPS和IRS的interneurons不是内生的振荡器，而他们的交流活动是由交互抑制9驱动。由于这些的interneurons直接抑制游泳足运动神经元，交替的PS-RS机芯生成10。运动然而，这不仅需要活动的产生，同时也协调不同的独立微电路。在游泳足系统，这种协调是通过协调微电路可确保四肢活跃在正确的时间建立。此微电路由三个识别神经元中的每个段11-15构建的。 该协议规定，日Ë首次一步一步夹层导向分离神经节（T4至A6， 图1）的链。我们展示了如何针隔离腹部神经索和desheathe每节。在这种孤立的神经系统的准备，负责游泳足的运动神经元是准备在电生理及形态学实验。此协议的第二部分展示了游泳足运动模式的主要特点。这包括了一步一步的指导，细胞外记录的游泳足的运动神经元的活动。 RS运动神经元的轴突通过神经N1的前支伸出，而PS的运动神经元的轴突通过同一神经的后支（ 图1）突出4。因此它们的活性可以从这些分支具有差分针电极进行记录。 <img alt="图1" fo:content-width="2in" src="/files/ftp_upload/52109/52109fig1highres.jpg" width="200" /><br/&gt; 图1:隔离从胸神经节4（T 4），以腹神经节6（A6）和它 T4 的示意图神经系统:胸神经节4; T5:胸神经节5; A1，A2 ...... A6腹神经节1，腹神经节2 ...腹神经节6; N1:神经N1; N2:神经N2; N3:神经N3; PS:电源冲程; RS:返回行程。定向缩写: A =前; P =后路。 这解剖程序和电技术展示是方便本科生和可能的生理补充学生实践课程。神经节的分离链已被用在大量的实验以研究神经系统的功能，协调性，或游泳足微电路6的调制以及在运动16,17适应性行为的神经元控制，小龙虾游泳足系统因此提供了一个巨大的量有趣的教学或T下雨，所有开始与小龙虾和假想马达图案的细胞外记录的腹神经索的解剖机会。

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Name of Material/ Equipment</td>
			<td style="width:131px;">Type</td>
			<td style="width:139px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:103px;">Comments/&#160;&#160; Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">4-channel extracellular amplifier: MA 102&#160;</td>
			<td style="width:131px;">Amplifier</td>
			<td style="width:139px;">Elektroniklabor, Zoologie, Universit&#228;t zu K&#246;ln, Germany</td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:131px;">air-table</td>
			<td style="width:131px;"></td>
			<td style="width:139px;">Technical Manufacturing Corporation 
			(TMC) a unit of AMETEK Ultra Precision Technologies, Peabody, MA, USA</td>
			<td style="width:103px;">63-534</td>
			<td style="width:103px;">for intracellular recording</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:131px;">Axon Digidata 1440A</td>
			<td style="width:131px;">Digitizer</td>
			<td style="width:139px;">Axon Instruments, Molecular Devices Design, Union City, CA</td>
			<td style="width:103px;">DD1440A</td>
			<td style="width:103px;">digitizes recorded signals&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">big bucket&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">filled with ice</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">Clampex &#38; Clampfit</td>
			<td style="width:131px;">pClamp 10, recording and analysis software</td>
			<td style="width:139px;">Molecular Devices Design, Union City, CA</td>
			<td style="width:103px;">pClamps 10 Standard</td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">cold lamp source</td>
			<td style="width:131px;">with flexible light guide (fiber optic bundle)&#160;</td>
			<td style="width:139px;">Euromex microscopes holland, Arnhem, BD</td>
			<td style="width:103px;">LE.5211 &#38; LE.5235</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">computer and monitor</td>
			<td style="width:131px;">equipped with recording software</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">container and pipette for liquid waste&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:131px;">crayfish saline&#160;</td>
			<td style="width:131px;">contains (in mM): 5.4 KCl, 2.6 MgCl2, 13.5 CaCl2, and 195 NaCl, buffered with 10mM Tris base and 4.7mM maleic acid; aerated for 3 hours. Adjust at pH of 7.4.&#160;</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">always keep at temperatures ~ 4&#176; C</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">dextran, Texas Red (3000MW, lysine fixable)</td>
			<td style="width:131px;">fluorescent dye, lysine fixable</td>
			<td style="width:139px;">Life Technologies GmbH, Darmstadt, Germany</td>
			<td style="width:103px;">D3328</td>
			<td style="width:103px;">for intracellular dyefill of neurons</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">differential pin electrodes</td>
			<td style="width:131px;">made from stainless steel &#632; 0.2 mm</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">dissection dish&#160;</td>
			<td style="width:131px;">(l x w x h) 15x7x5 cm; linned with black silicone</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">used in the gross disection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">faraday cage</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">fixing pins</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for pinning the specimen</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">forceps (biology, Dumont #5)</td>
			<td style="width:131px;">Forceps: Biology, tip 0.05 x 0.02 mm, length 11cm, INOX</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">11252-20</td>
			<td style="width:103px;">fine forceps: used to pick nerves</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">forceps (biology, Dumont #55)</td>
			<td style="width:131px;">Forceps: Biology, tip 0.05 x 0.02 mm, length 11cm, INOX</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">11255-20</td>
			<td style="width:103px;">extra fine forceps: used for desheathing</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:131px;">forceps (electronic, Dumont #5)</td>
			<td style="width:131px;">Forceps: Standard, tip 0.1 x 0.06 mm, length 11cm, INOX</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">11251-20</td>
			<td style="width:103px;">coarse forceps:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; used to grab specimen and pins</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:131px;">intracellular electrode</td>
			<td style="width:131px;">Borosilicate glass capillaries (outer/inner diameter: 1mm/0.5mm), with filament</td>
			<td style="width:139px;">Sutter Instruments, Novato, CA</td>
			<td style="width:103px;">BF100-50-10</td>
			<td style="width:103px;">for intracellular recording and dyefill of neurons</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">Leica S8 Apo StereoZoom</td>
			<td style="width:131px;">Dissection Microscope&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Zoom 1x - 8x</td>
			<td style="width:139px;">Leica, Germany</td>
			<td style="width:103px;">10446298</td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">microscope table</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">mirror</td>
			<td style="width:131px;">to illuminate preparation from below</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">modeling clay</td>
			<td style="width:131px;"></td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">Olympus SZ61</td>
			<td style="width:131px;">Dissection Microscope&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Zoom 0.67x - 4.5x</td>
			<td style="width:139px;">Olympus, Germany</td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for the dissection</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">petri dish&#160;</td>
			<td style="width:131px;">94 x 16 mm; lined with clear silicone</td>
			<td style="width:139px;">Greiner bio-one, Germany</td>
			<td style="width:103px;">633180</td>
			<td style="width:103px;">used to pin the isolated chain of ganglia</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:131px;">ring scissors</td>
			<td style="width:131px;">ThoughCut, cutting edge: sharp/blunt, straight: 13cm</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">14054-13</td>
			<td style="width:103px;">for gross dissection&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (steps 2.1 - 2.11)</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:131px;">saline dispenser&#160; with a 16 gauge needle (outer &#632; 1.6mm) attached via a flexible tube.</td>
			<td style="width:131px;">Volume ~ 60ml,</td>
			<td style="width:139px;"></td>
			<td style="width:103px;"></td>
			<td style="width:103px;">used for exsanguination</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:131px;">spring scissors or alternative: Vannas spring scissors</td>
			<td style="width:131px;">cutting edge: 8 mm, tip diameter: 0.2mm, straight: 10cm or cutting edge 2.5 mm, tip diameter 0.075 mm, straight: 8cm</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">15024-10 or&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 15000-08</td>
			<td style="width:103px;">for desheathing&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:131px;">stainless steel wire &#632; 0.125 mm&#160;</td>
			<td style="width:131px;">to cut pins of 4-7 mm length</td>
			<td style="width:139px;">Goodfellow GmbH, Bad Nauheim, Germany</td>
			<td style="width:103px;"></td>
			<td style="width:103px;">for pinning of the nerve cord</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:131px;">student Vannas&#160; spring scissors or alternative:&#160; Moria Spring Scissors</td>
			<td style="width:131px;">cutting edge: 5mm, tip diameter: 0.35mm, straight: 9cm or cutting edge: 5mm, tip diameter 0,1 mm, straight: 8 cm</td>
			<td style="width:139px;">Fine Science Tools (FST), Germany</td>
			<td style="width:103px;">91500-09 or&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 15396-00</td>
			<td style="width:103px;">for gross and fine disection&#160;&#160;&#160;&#160;&#160;&#160;&#160; (steps 2.11 - 3.14)</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:131px;">sylgard</td>
			<td style="width:131px;">184 Silicone Elastomer Base and Curing Agent; for black sylgard add activated carbon</td>
			<td style="width:139px;">Dow Corning, Midland, MI, USA</td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:131px;">syringe filled with petroleum jelly and equipped with a 20 gauche needle with rounded tip</td>
			<td></td>
			<td></td>
			<td></td>
			<td style="width:103px;">for extracellular recording</td>
		</tr>
	</tbody>
</table>

the swimmeret system of crayfish: a practical guide for the dissection of the nerve cord and extracellular recordings of the motor pattern

在这里，我们证明小龙虾腹部神经索的解剖。该制剂包含的最后两个胸神经节（T4，T5）和腹腔神经节的链（A1到A6）。这种链节包括中枢神经系统（CNS），其驱动所述腹肢（游泳足）的协调运动的部分:游泳足系统。它是已知的，在小龙虾每个游泳足由它自己的独立模式生成内核生成节奏交替活动1-3从动超过五十年。运动神经元支配每个游泳足的肌肉组织包括两个解剖学和功能上不同的人群4。一个是负责对游泳足的缩回（动力行程，PS）。其他驱动游泳足的前伸（回击，RS）。所述游泳足系统的运动神经元都能够自发产生假想马达模式，这是相同的记录在体内图案</EM&gt; 1。 本报告的目的是介绍一个有趣和方便的模型系统研究的节奏生成网络和协调学生的实践课程，实验室独立的微电路。所提供的协议包括一步一步的说明小龙虾的腹神经索的解剖，神经节钉扎的孤立链，desheathing的神经节，并从隔离神经系统记录游泳足虚构的运动模式细胞外。 此外，我们还可以监视从树突细胞内记录的游泳足的神经元的活性。在这里，我们还简要介绍这些技术，并提供了一​​些例子。此外，游泳足神经元的形态，可以使用各种染色技术进行评估。在这里，我们提供的细胞内（通过离子电渗疗法）染料填充神经元和游泳足运动神经元的池的回填的例子。在我们的实验室我们使用这种制剂来研究微电路之间假想运动，感觉反馈对CNS的活性的效果，和协调的基本功能在细胞水平上。

与从RS和PS，1神经节的运动神经元的同时外记录，这些运动神经元池的交替活性，可以监测（ 图18）中，表示假想运动图案。 <img alt="图18" src="/files/ftp_upload/52109/52109fig18highres.jpg" /> 图18:示意图1节和差分引脚电极的放置 RS运动神经元的细胞外记录（上部曲线）和PS运动神经元（下迹线）。 同侧PS 2〜PS 5运动神经元的胞 ​​外PS活动突出了节间的协调，在游泳足系统（ 图19A），有趣的方面。 首先，活动总是与A5（ 图19A， 第 4 次的迹线）是PS脉冲串开始，并在前部方向上的激励波传播（&lt;STRONG&gt;图19A，绿线）。第二，在期间（也就是从一个脉冲串的开始测量到发病的下一个脉冲串的时间）在各段保持恒定（ 图19A，青色箭头）。这是真实的，只要从一个神经节到下一个信息流是完整的，并在实验条件不会改变。 在不同的制剂或不同的实验条件下的期间和脉冲串持续时间可以变化基本上:从0.25周期为1秒。为了不同制剂之间比较这些参数，它们需要被归一化并且绘制在相直方图（ 图19B）。这揭示了马达图案的第三有趣的方面:​​段之间的相位滞后，在段间延迟，保持不变和独立的节奏的频率。 来计算相位的直方图，以下的测量和计算是necessary: 首先，将参考曲线必须选择。通常我们使用最后神经节的聚苯乙烯微量（ 例如 ，PS 5）作为基准（即时间为0），用于在多个段（ 图19A）。然后记录在游泳足节奏，测量的周期的相位的直方图（以毫秒为）从一个PS 5突发（时间0开头的节奏， 图19A，橙色点线）到下一个的PS 5突发（ 图19A，青色箭头）.Within此周期的开始，测量延迟（以毫秒为）在PS 5突发（时间0）和起始（突发发作， 图19A和B，橙色箭头）和结束的开始之间（偏移图19A和B，紫罗兰箭头）的PS突发中的每个其他神经节。因此，为了计算相位发作和偏移对每个脉冲串的延时是由并行段划分。这些测量将导致数字V0和1之间alues ​​最后，在一个相位的直方图， 如图19B绘制这些值。 所得相位的直方图（ 图19B）包含有关对PS的脉冲串在每个段中的占空比信息。和0.25的〜相邻段之间的节间相位滞后是清晰可见。 <img alt="图19" src="/files/ftp_upload/52109/52109fig19highres.jpg" /> 图19:（A）电极放置和同侧的PS 5至所需的相位的直方图的PS 2（B）中的测量和计算的细胞外记录的示意图。从连续10脉冲串（N = 10）中计算出的相位的直方图显示了〜0.25的节间的相位延迟。 对于更高级的实验者，细胞内的录音与运动神经元的树突急剧微电极可以是有用的。该膜电位个别的细胞内记录的PS的运动神经元显示出同相振荡与外记录活动同一hemiganglion的所有的PS的运动神经元（ 图20中 ，灰色图）。可替换地，RS运动神经元或的interneurons可以在细胞内以相同的方式记录的（数据未示出）。 <img alt="图20" src="/files/ftp_upload/52109/52109fig20highres.jpg" /> 图20:电极位置的示意图的一个细胞内记录的PS运动神经元（上跟踪）的膜电位振荡阶段（灰色面板）与细胞外记录的PS活动（下迹线）。 对于识别细胞内记录的神经元（或的interneurons运动神经元）的，细胞可使用锋利的微电极和电离子透入（见讨论）染料填充。在图21中 ，2细胞内染料填充PS的运动神经元被示出。其腹胞体位于后方的神经N1的基极。初级神经突的项目前方（ 图21B-2）和分支横向神经纤维（ 图21A和B-3）之内;通过神经N1的后支到游泳足肌肉（ 图21B-4）中轴突的项目。 <img alt="图21" src="/files/ftp_upload/52109/52109fig21highres.jpg" /> 图21:腹部神经节（A）原理图（B）两种细胞内染色PS运动神经元。 （1）胞体; （2）初级突起; （3）树枝状分枝在横向神经毡; （4）轴突通过神经N1的后支的项目;栏= 100微米。 对于欠发达的实验者，或谁想要学习腹神经节的形态，从通过神经N1的PS和RS运动神经元池回填，是一个有趣的练习。在图2中2中，RS和1 hemiganglion PS的运动神经元染色用Co 2+（RS）和Ni 2+离子（PS），分别。胞体的PS的运动神经元位于后方的神经N1（ 图22-1）的基极。的RS的运动神经元的所有胞体均位于前向神经N1（ 图22-2）的基极。 <img alt="图22" src="/files/ftp_upload/52109/52109fig22highres.jpg" /> 图22:从回填前（橙色， 钴离子），后（蓝，镍离子 ）N1神经分支 （1）PS运动神经元胞体; （2）胞体RS运动神经元。栏= 100微米。 <img alt="图3" src="/files/ftp_upload/52109/52109fig3highres.jpg" /> 图3:去除所述爪（1）和腹足（2）。红线（1）和（2）表明削减头寸。棒= 5厘米。 图4:（A）的动物被钉扎腹侧和固定在尾节（B）中的小龙虾通过爪开口用冷盐水放血。棒= 5厘米。 <img alt="图5" src="/files/ftp_upload/52109/52109fig5highres.jpg" /> 图5:（A）斩首和（B）去除腿走路的。红色线条表示削减岗位。酒吧= 1厘米。 <img alt="图6" src="/files/ftp_upload/52109/52109fig6highres.jpg" /> 图6:（A，B，C和D）去除的前部和头胸部的横向部件。沿外侧腹部E.开幕。红线（1），（2），（4），（5）表示削减岗位。 （3）消化腺。红色箭头指向已经打开腹腔。酒吧= 1厘米。 <img alt="图7" src="/files/ftp_upload/52109/52109fig7highres.jpg" /> 图7:（A）将试样抓起在行走的腿（红色箭头）的开口（B）的背侧肌肉股线（1）的同时，检体被打开横切。白色箭头示出了在其中胸骨板被拉出的方向。腹腔（C）的概述与胸骨动脉（2）和神经索（3）。 <img alt="图8" src="/files/ftp_upload/52109/52109fig8highres.jpg" /> 图8:小龙虾腹部固定背肌肉股（1）横向视图和胸骨动脉（2）;红线（3）和（4）表示削减岗位。酒吧= 5毫米。 <img alt="图9" src="/files/ftp_upload/52109/52109fig9highres.jpg" /> 图9:背和腹部的腹侧部分彼此分离 （1）红色线表示切割的位置。胸骨板（2）与腹神经索;背腹部（3）的一部分。 <img alt="图10" src="/files/ftp_upload/52109/52109fig10highres.jpg" /> 图10:（A）中的镊子的位置以去除前cephalothoracic胸骨（B）的红色线（1）和（2）指示在何处切断剩余cephalothoracic胸骨之间的肌肉（C）的胸神经被横断处红色线（3），以隔离从周围组织的胸神经节。红线（4）表示T3和T4之间的切口。酒吧= 5毫米。 图11:（A和B）查看胸骨板在A2的。神经N1被从所述组织中分离出与沿后胸骨表皮内折（1）和前两者胸骨表皮infoldings（B 2）的切口（C）的高倍围绕A2中的区域，与分离的神经N1。（ D）隔离在对侧类似的削减神经N1的。红线（2）和（3）表示削减职位; （1）前，后胸骨表皮infoldings;酒吧= 5毫米。 <img alt="图12" src="/files/ftp_upload/52109/52109fig12highres.jpg" /> 图12:（A ​​和B）的A6从组织中分离和神经索可在A6的神经解除（℃）。 </s仲&gt;腹部神经节向腹侧腹部大动脉所有连接都横断。（D）的分离的神经索转移到培养皿中衬有明确SYLGARD 和填充有生理盐水。红色线条表示削减岗位。酒吧= 5毫米。 <img alt="图13" src="/files/ftp_upload/52109/52109fig13highres.jpg" /> 图13:1节识别的神经索的背，腹侧（黑线） 的横向视图 。巴=5毫米<img alt="图14" src="/files/ftp_upload/52109/52109fig14highres.jpg" /> 图14:神经N1是从后支栏=5毫米分离的前支 。 <img alt="图15" src="/files/ftp_upload/52109/52109fig15highres.jpg" /> 图15:方向为第一小切口的腹部神经节（A）通过连接词（红色箭头）的护套位置的desheathing，放在外侧和后侧的神经节。通过连通上述套管内的后续切割的标志是红线（1）（B）红色线（1）和（2）标记沿着神经节的横向边界的切割。护套可以事后从神经节拉动。酒吧总数= 1毫米。 <img alt="图16" src="/files/ftp_upload/52109/52109fig16highres.jpg" /> 图16:录音设置（一）概述（二）材料和所需的细胞外记录的工具。 C.设备所必需的电生理记录。 （1）冷灯源; （2）法拉第笼; （3）体视显微镜; （4）气表; （5）显微镜表; （6）镜; （7）差分引脚电极; （8）注射器填充有石油果冻; （9）橡皮泥; （10）镊子; （11）液体废物处理; （12）数字化; （13）外的放大器; （14）电脑，配备了录音软件和显示器。 <img alt="图17" src="/files/ftp_upload/52109/52109fig17highres.jpg" /> 图17:（A和B）记录（1），参考（2）脚电极的位置。 （C和D）的神经的后支N1为弯曲所述记录电极周围。记录电极E的底座（4）分离为具有凡士林。 F的完整记录电极分离为具有从浴溶液凡士林。 （3）注射器填充有凡士林。酒吧= 2毫米。

Watch this Scientific Journal Video about The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern at JoVE.com

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

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 ...

the-swimmeret-system-crayfish-practical-guide-for-dissection-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.0K 次观看.  University of Cologne. 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. 

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

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 ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.