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>

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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...

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...

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

Research

JoVE Journal

Neuroscience

11.9K 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

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

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 ...

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 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 ...

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.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

你在哪里切的问题: 眼地标性小鼠视网膜空间定位的解剖与分析指南

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...