Name: an in vitro adult mouse muscle-nerve preparation for studying the firing properties of muscle afferents
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This work was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant (KAW), a grant from Pfizer (WS753098, SH), and an NIH MARC fellowship (#2T34GM008253-21, JAF). The authors would like to thank Michael Sawchuk, Anusha Allawala, Nina Bubalo, Remie Mandawe and Peter Nguyen for their technical support.

适当的国家和机构伦理应该进行动物实验前获得。 1，拆卸EDL肌肉和神经的<ol><li>称量并深深麻醉成年小鼠吸入异氟烷用5％异氟醚蒸发器加上1.5升/分钟的氧气流量或钟形罩，底部异氟醚浸泡的棉花。 </li><li>确保鼠标是深度麻醉，不给脚趾捏回应。快速斩首使用大，锋利的剪刀或断头台。 </li><li>请通过用剪刀肋骨下腹正中切口和除去内脏。 </li><li>通过抓住从颈部区域的皮肤和拉过去脚部皮肤的动物。 </li><li>通过切割上述的臀部除去腿部和放置面皮腿与冷藏（4℃）的培养皿，carbogenated（95％O 2，5％CO 2）低钙，高镁的碳酸氢盐缓冲的盐水溶液周转箱宁以mM:128氯化钠，氯化钾1.9，1.2 KH 2 PO 4，26的NaHCO 3，0.85氯化钙 ，16 6.5 MgSO 4干燥，和10葡萄糖（7.4±0.05的pH）。低钙，高镁溶液在解剖抑制突触传递。 </li><li>将腿背侧的菜和脚的腿和臀部向下用针或昆虫针，使得膝关节和踝关节是在一个90°的角。放置在脚的每一端与一个销上的每个前侧和后侧的大腿的销来固定到位的组织。 </li><li>使用Castroviejo弹簧剪刀，抬起肌的大腿上的顶层，并切割以暴露坐骨神经直接下方的中线。坐骨神经位于正下方的顶端肌肉层和运行从其出口股骨附近的臀部上方，直到其分支成刚膝关节前腓总和胫神经。 </li><li>删除上述坐骨神经肌肉，直到POINT在此深腓神经的分支潜入拇长屈肌（FHL）肌肉可见。 </li><li>使用＃55钳子，解剖周围腓分支的结缔组织，从腓肠肌和比目鱼肌，这是对内侧胫骨释放它。切断腓肠肌和比目鱼肌的肌腱和小心地从神经下将其删除。 </li><li>除去在胫骨的侧面浅表肌肉以暴露三个不同的腱束在踝关节，从内侧到外侧:FHL，EDL，和胫骨前肌（TA），与EDL隐藏在TA下方。 </li><li>切助教肌腱在踝关节，从EDL解除助教起来了，并切断肌肉膝盖附近将其删除。 </li><li>切FHL肌腱在脚踝，将其返回到到达那里的神经进入FHL的区域。切略低于该点并删除FHL肌肉〜2/3。 </li><li>切断坐骨神经尽可能靠近髋关节为POSsible，轻轻去掉所有的神经分支除外腓深神经分支。 </li><li>切筋EDL在两个用大弹簧剪刀踝关节和膝关节。 </li><li>使用锋利的剪刀，从周围组织中取出的EDL，剩余FHL和神经通过在膝关节切削穿过胫骨和中途的大腿。切去其余的胫骨，这样就在EDL中，信望爱和神经的一部分保留下来。 </li></ol> 2，安装EDL肌肉和神经进入组织浴（图1A） <ol><li>在实验开始之前，准备的组织浴中。不断灌注同浴含氧（100％O 2）合成的组织间液（SIF）含（单位mm）123氯化钠，氯化钾3.5，0.7 硫酸镁 ，1.7 磷酸二氢钠，2.0 氯化钙 ，9.5 NAC 6 H 11 O（葡糖酸钠），5.5葡萄糖，7.5蔗糖和10 N -2 - hydroxyethylpiperazine- N&#39;-2 -等hanesulfonic磺酸（HEPES）; pH值7.4±0.05 17。 15-30毫升/分钟的流速被推荐。显示的是25毫升容量与固定在浴缸的底部，有一个组织安装后和安装的力和长度控制器（约计浴缸尺寸2刺激电极市售洗澡8.5厘米×3厘米×1厘米;适合的产品。参照表的材料/设备）。 </li><li>用剩余的FHL组织处理隔离肌肉神经，并将其放入组织浴。放置一小片SYLGARD对培养皿的底部，并用驱虫针通过剩余FHL组织稳定的肌肉。 </li><li>用6-0丝线缝合扎两个筋并加盖一端到组织后，其他的力和长度控制器的控制杆臂（见表材料/设备规范）。使用最小的缝线长度是可行的。取出昆虫针和SYLGARD你绑筋后。注:为便于连接到所述杠杆臂的小片金属丝可以弯曲成"J"形钩和固定到该杆臂与环氧树脂。缝合线可以被连接到导线，而不是拧入于该杆臂的小孔。 </li><li>请从SA的16玻璃吸电极先用玻璃微拉马（热火= 286，拉= 0，速度= 150，时间= 200）;突破尖端背部和手动磨它放在磨刀石，直到有大约3毫米的锥形。使用microforge尖端融化到10之间所需的尖端内径- 100微米取决于神经的一个希望从取样的区域（ 见图1B，1C的电极原理图和表格材料/设备产品信息）。 </li><li>填写预制玻璃吸电极的内银线与SIF。 </li><li>抽吸神经的切断端插入电极和连接到差分放大器的正端口。包裹在电极与氯化的银导线连接到探头的负极端口。从浴缸到探头的接地端口上运行的第二氯化的银线接地的SIF洗澡。另外地灌注管法拉第笼在多个点，以减轻通过灌注泵推出的电噪声。 </li><li>刺激通过安装在该组织浴中的肌肉的任一侧以诱导肌收缩，​​电极的肌肉。可选地放置在神经的切断端部的刺激电极。增加刺激电压，直到峰值收缩力观察，然后增加电压通过一个额外的15％，达到超强的电压（0.5毫秒脉冲宽度）。继续抽搐收缩在超最大电压，用10秒的休息在中间，但改变肌肉的长度，直到峰值收缩力达到找到肌肉的最佳长度（L O）。所有的长坡道和震动将开始与肌肉在这个冷日。 </li><li>允许肌肉的神经制剂保持在该浴中的至少1小时，随后的数据采集之前，以允许所述组织，以达到熔池温度和正常的突触传递，以恢复以下清扫低钙溶液。 </li><li>为了收集数据，在比室温等的温度，将温度探头到附近的肌肉组织浴。慢慢地由穿过组织浴底板泵送加热的水使浴加热到所需温度。环绕SIF水库粘土微波炉加热垫，以帮助保持稳定的温度。 </li></ol> 3，数据收集<ol><li>要确定一个传入的梭传入，在一个0.5毫秒超强电压刺激产生重复发表抽搐收缩每秒一次记录的神经元活动。注:肌梭传入应抽搐收缩18,19（ 图2）期间暂停。 </li><li>使用DATA采集软件适用长度的变化以不同的速度和不同的长度。使用自定义脚本来自动完成这个任务（见补充信息的脚本和方向如何自定义给出的延伸截图）。应用4秒的斜坡和保持伸展为2.5％，5％，7.5％L O伸缩长度为20，40，或60％L O / 15秒的拉伸速度。注:请参阅具体的力量和长度控制器所需的电压 - 毫米换算系数使用手册。 </li><li>在实验结束时，确定肌肉健康在24℃下使用最大等长强直收缩（500毫秒列车，120赫兹串频率，0.5毫秒的脉冲宽度，超最大电压）。峰值收缩力比较先前报道的值（〜24牛顿/厘米2 9,20）。 </li></ol>

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The open access charges for this manuscript have been paid by Aurora Scientific, Inc., the company that makes the force and length controller and in vitro bath plate used in this manuscript.

这篇文章的目的是描述了一种从肌梭传入在一个孤立的小鼠肌肉，神经的准备记录。我们已经发现，小鼠梭传入响应类似于伸展那些从大鼠，猫和人15，和其它的实验室已经使用小鼠作为模式生物研究中均肌肉和体外的皮肤感觉神经元（例如3,13） 。 肌肉感觉传入可以在两个24°C和34°C的记录至少6-8小时。以最小化处理的EDL和神经，使用FHL的剩余部分，​​以处理组织只要有可能。以下的夹层，等待至少1小时，以启动数据收集，以使组织平衡到浴温度和正常的突触传递，以进行恢复。先前，肌肉健康是在此制备中所用的肌肉的子集，通过确定最大强直禁忌验证由肌肉产生的ctile力是类似的报告由他人在实验15的开始和结束。 肌梭传入进行功能识别在该制备通过在烧制响应于抽搐收缩（ 图2）寻找一个特性停顿以及预期的瞬时频率增加响应长度的变化（ 图3）。根据我们的经验，大多数传入那团火在控制条件是肌梭传入。保留在该制剂（〜7毫米最大）的神经的长度不够长以使用传入传导速度的差异，以确定肌肉传入神经 ​​亚型13。此外，不像猫和人类的动态灵敏度措施未能明确区分组IA和II传入小鼠（进一步讨论见威尔金森等人。15）。传入的其他亚型（ 即，，第III族和第IV）添加物质，如辣椒辣素，ATP，或缓激肽，降低染液的pH，暴露肌肉局部缺血等 3,13,21-23在此制备使用附加的功能测试可以被识别，例如使用抽吸电极可以从多个感觉神经元的活动被记录一次，这增加了数据，这些数据可以从一个单一的肌肉被收集量。该制剂可用于衡量对感觉神经元群体的反应或识别传入的响应扰动的整体效果。如果尖峰形状是独特的是，高达4的感觉神经元可以通过软件进行鉴别（两者的Spike2（剑桥电子设计）和LabChart临（AD仪表）也同样进行）。在神经细胞不能够被辨别的情况下，改变在电极放置或尖端直径可以很容易地实现。 总之，鼠标肌肉神经体外 preparATION是一个简单的实验方法可用于研究的肌肉感觉传入的响应特性，以各种物理化学的干扰，损伤和疾病模型。此外，该制剂特别适合于利用在小鼠中可用的功能强大的遗传工具，包括转基因动物和光遗传学工具的优点。

骨骼肌是由感觉神经元提供对中枢神经系统具有对肌肉环境的重要信息支配。肌梭是高度专业化的梭内肌纤维分布在梭外肌纤维平行的由封装结构。主轴是由集团IA和II传入编码改变肌肉的长度和梭内纤维音通过动态伽玛支配运动神经元1规定支配。 Ib族传入位于肌纤维和它们的肌腱插入之间，并且在肌肉2收缩的变化，肌肉力量，例如敏感。第Ia，Ib和II传入提供有助于适当的电机控制本体的反馈。位于整个肌肉肌肉传入的附加种群（组III和IV），用于发信号的代谢物的积累，伤害性刺激的存在下，和肌肉温度<sup&gt; 3。此肌肉感觉信息是维持体内平衡，防止肌肉损伤和调节运动的关键。肌肉的传入可以根据以往的经验4改变他们的射击模式。伤害感受器的活化可导致慢性疼痛状态5和从肌肉本体感受器异常信号的诱导可导致问题的平衡或运动6。我们使用的分离的肌肉神经的体外制备，研究肌肉感觉神经元受体末梢从两种性别的成年小鼠的响应（示出的是从2-4个月大的C57BL / 6小鼠的响应）。鼠标是模型的遗传物质在哺乳动物的研究。这种准备需要坐骨神经腓深支和趾长伸肌（EDL）的肌肉，小腿外侧部发现腓骨组的快肌的隔离。该EDL经常用于研究肌肉收缩特性7-9，具有德ndons，很容易分离和足够小，以允许足够的扩散供氧在休息和合理的收缩工作周期10。其他的肌肉在这个区域（例如比目鱼肌和胫骨前肌）具有类似尺寸的和该制剂可以很容易地修改从这些肌肉传入记录。一吸入电极被放置在神经记录肌肉感觉传入神经烧成的切断端。单个神经元可被识别和分析的基础上使用穗排序软件的尖峰形状。刺激在洗澡或对神经的电极可用于唤起肌肉收缩。肌肉长度和力可以被控制，并使用市售的系统测得的。类似体外制剂已被用于在啮齿动物研究III族和第IV肌肉传入在大鼠EDL 11，Ia族和II梭传入在大鼠第四蚓状趾肌12和III族和第IV肌肉传入的叔他鼠标跖肌13。 一种体外系统具有药理访问和直接控制如温度和pH值灌洗液和物理变量的优点。 体外方法消除体内的潜在混淆的麻醉和肌肉的灌注状态。而肌肉的神经制剂允许的扰动对传入的直接反应的研究中，研究主轴敏感度等综合应对伽玛传出调制的能力是可以在体内14或离体3准备的牺牲有这个准备没有神经元胞体。 这种准备是以前用来表征鼠标肌梭传入到斜坡的电池反应并保持伸展和震动，它被确定鼠标梭传入responsES类似于所报告的其他物种例如大鼠，猫和人类。鼠标梭传入的反应被认为是在两个24°C和34℃的水浴温度相似，虽然在34℃的绝对发射率和更快的传入更能够更快地响应长度变化15。我们在下面说明如何编写可用于确定和研究的肌梭传入。此外，该制剂可以很容易地进行修改，以研究其他肌肉传入神经 ​​亚型13的响应，以响应于药物或疾病状态或分类的其他变量（ 例如年龄，性别，基因敲除）比较感觉传入的属性。

<table><tbody><tr><td>Force and Length Controller &amp;amp; Tissue Bath</td><td>Aurora Scientific, Inc.</td><td>300C-LR &amp;amp; 809B-IV&amp;nbsp;</td><td></td></tr><tr><td>Masterflex L/S Digital Drive Perfusion Pump &amp;amp; Easy Load II Pump Head</td><td>Cole Parmer</td><td>EW-07523-80 &amp;amp; EW-77200-60</td><td></td></tr><tr><td>2 Channel Microelectrode AC Differential Amplifier &amp;amp; Headstage Amplifier</td><td>A-M Systems</td><td>Model 1800; 700000 &amp;amp; 700500</td><td></td></tr><tr><td>Simulator</td><td>Grass&lt;br/&gt; OR&lt;br/&gt; Aurora Scientific, Inc.</td><td>S88&lt;br/&gt; OR&lt;br/&gt; 701 C</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Swift</td><td>892070</td><td></td></tr><tr><td>Fiber Optic Light Source</td><td>Fiberlite</td><td>Model 190</td><td></td></tr><tr><td>Analog to Digital Board</td><td>AD Instruments</td><td>PL3508/P (PowerLab 8/35)</td><td></td></tr><tr><td>Data Acquisition &amp;amp; Analysis Software</td><td>AD Instruments</td><td>LabChart Pro 7</td><td></td></tr><tr><td>Electrode Holder</td><td>A-M Systems</td><td>672445</td><td></td></tr><tr><td>Electrode Glass</td><td>Dagan</td><td>SA16</td><td></td></tr><tr><td>Electrode Puller</td><td>Sutter Instrument Co.</td><td>P-80/PC</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-9</td><td></td></tr><tr><td>Isoflourane</td><td>MWI Veterinary Supply</td><td>18627</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Surgical Tools&lt;/strong&gt;</td></tr><tr><td>Large Dissecting Scissors</td><td>Fine Science Tools</td><td>14014-17</td><td></td></tr><tr><td>Large Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Large Spring Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>#5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>#55 Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Sharp Scissors</td><td>Fine Science Tools</td><td>14059-11</td><td></td></tr><tr><td>6-0 Silk Suture</td><td>Fine Science Tools</td><td>18020-50</td><td></td></tr><tr><td>Chemicals for Low Calcium- High Magnesium aCSF and SIF&lt;br/&gt; (Low Calcium solution is equilibriated with 95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt; gas and pH of 7.4 &amp;plusmn; 0.05; SIF is equilibriated with 100% O2 gas and pH of 7.4 &amp;plusmn; 0.05)</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888-1KG</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>230391-500G</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>223506-500G</td><td></td></tr><tr><td>Sodium gluconate</td><td>Sigma</td><td>S2054-500G</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S8282-500G</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767-500G</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S5016-500G</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-250G</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P0662-500G</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P3911-500G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6014-500G</td><td></td></tr></tbody></table>

an in vitro adult mouse muscle-nerve preparation for studying the firing properties of muscle afferents

肌肉感觉神经支配肌梭和高尔基腱器官编码的长度和力量的变化必需的本体。额外传入纤维监视其他特征肌肉环境，包括代谢物的积累，温度，和感受伤害的刺激。总体而言，感觉神经元的异常激活可能导致运动障碍或慢性疼痛综合症。我们描述趾长伸肌（EDL）的肌肉和神经的，在成年小鼠拉伸诱发传入的反应在体外研究中的隔离。感官的活动记录从神经与抽吸电极和个人传入可以用秒杀排序软件进行分析。 体外准备允许在感觉传入了很好的控制研究没有麻醉的潜在的困惑或改变肌肉的灌注。在这里，我们描述了一个协议来识别和测试肌梭传入响应舒展。重要的是，这种准备还支持肌肉的传入，响应特性下列药物的应用和强大的遗传方式和疾病模型小鼠中掺入其他亚型的研究。

肌肉传入的反应可以下面的各种扰动被记录，这取决于其传入亚型正在研究上。肌梭传入肌肉收缩和坡道和保持弹力代表答复如下图所示。 图2来识别传入的梭传入，抽搐收缩给出每秒一次（0.5毫秒脉冲宽度），看看是否有在收缩18,19烧成一个暂停。显示的神经元活动和肌肉紧张时代表跟踪这些肌收缩。抽搐时不收缩的神经元活动时观察到，如预期的梭传入。如果所记录的传入是Ib族高尔基腱器官传入，增加了收缩时的燃烧速率将期望。 在控制条件下最传入一个普通的放电模式（〜12脉冲/秒，在24℃〜〜32脉冲/秒，在34℃下）是肌梭传入。梭传入的一个子集，只会火拉伸过程中（在我们的手中〜梭传入的11％）15。 图3A显示了两种肌梭传入具有代表性的原始跟踪响应斜坡和保持受力和长度控制器产生的弹力。 LabChart的峰值直方图功能是用来识别和分析两个单个神经元的瞬时发射频率分开（ 图3B-3C）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig1highres.jpg" width="500" /> 图1:隔离肌肉制备A）趾长伸肌（EDL）和支配坐骨神经被安装在一个孤立的组织浴灌注含氧合成间质液（SIF）。细胞外放大器连接到sucti对电极记录神经活动。双力和长度控制器，用适当的软件控制和措施的肌肉力量和长度。组织浴电极提供刺激来产生抽动或强直收缩。通过内部浴盘频道抽热水控制组织浴温度。 二）拉玻璃吸电极的理想针尖形状，因为这是用microforge产生的吸力电极〜3毫米锥形尖端，C）的放大写照。 <a href="https://www.jove.com/files/ftp_upload/51948/51948fig1highres.jpg" target="_blank">请点击这里查看本图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig2highres.jpg" width="500" /> 图2主轴传入响应抽搐收缩。神经activitY（上图）和30抽搐的肌肉收缩张力（底部的迹线）叠加在黑色的顶部重叠的痕迹。在收缩引起的张力增加，这是肌梭传入的特征响应传入活动暂停。收缩的过程中支架和箭头上面的顶部曲线表示的时候活动已暂停。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51948/51948fig3highres.jpg" width="600" /> 图3主轴传入响应斜坡期间斜坡和保持舒展）两个传入（上部曲线）原始神经活动和保持应用到EDL（在底部的迹线显示肌肉的长度）。 二）瞬时发射频率（研究所舒展。大一，对参展单位从A L O活动的每秒脉冲）（以蓝色显示）。 三）Instantane较小的单位项发射频率，在拉伸唯一火灾（图中橙色）。这两个单位的拉伸是肌梭传入1特征中表现出的秒杀频率调整。

Watch this Scientific Journal Video about An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents at JoVE.com

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

肌肉感觉神经元参与本体感受器的信号，并在代谢状态和伤害相关的事件报告。我们描述了成年小鼠的体外肌肉神经准备在拉伸激活肌肉传入的研究。

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

an-vitro-adult-mouse-muscle-nerve-preparation-for-studying-firing

Research

JoVE Journal

Neuroscience

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

17.0K 次观看.  San José State University. 肌肉感觉神经元参与本体感受器的信号，并在代谢状态和伤害相关的事件报告。我们描述了成年小鼠的体外肌肉神经准备在拉伸激活肌肉传入的研究。 

视频: An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

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 ...

科研

神经科学

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

同时细胞内记录的车组在成年小鼠的腰椎运动神经元产生的力<em在体内</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

医学

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

腓神经损伤方法:可靠检测，以确定和测试因素修复神经肌肉接头

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

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