这项研究得到了库鲁汶 ("开放未来" 和 C1), 科研基金 (FWO 住宿), 亨利 Latran 基金会, 杂色反疾病神经 Musculaires (ABMM), 肌肉萎缩症协会 (MDA), 和 als 协会和 als (比利时)。PVD 拥有 FWO-住宿的高级 investigatorship。RP 得到了来自中央治疗诊所 (CRC) 爱尔兰的赠款的支持, 目前得到爱尔兰国立大学和 FWO 的支持。

根据库鲁汶大学的指导方针和相关的欧洲准则 (欧洲联盟指令 2010/63/欧盟的动物实验), 所有动物都被安置在标准条件下。所有动物实验均由库鲁汶地方伦理委员会批准。
1. 动物制剂和麻醉
<ol><li>用异氟醚/氧吸入诱导小鼠麻醉。使用4% 的异氟醚诱导麻醉和2-3% 的维护在2.5 升/分钟的氧气流量。根据老鼠的情况, 调整异氟醚的麻醉维护率,即小鼠和弱老鼠所需的麻醉剂较少。确认适当的麻醉,例如, 对后肢步行垫施加轻度压力, 检查是否有疼痛戒断反射。</li>
	<li>用恒温加热板在37摄氏度控制小鼠体温, 防止麻醉时体温降低。</li>
	<li>将鼠标与 nosecone 配合以维护麻醉。确保动物有足够的氧气通过检查 nosecone 不阻断呼吸道和动物呼吸稳定。</li>
	<li>在录音过程中, 通过观察呼吸速度 (大约1赫兹麻醉) 和缺乏在轻度压力下的退缩反射来监测老鼠是否有足够的麻醉。如果麻醉不够深, 手动增加异氟醚浓度。</li>
	<li>测量后, 让鼠标在加热板或红外线灯的温暖状态下恢复, 直到它恢复了足够的意识维持胸骨卧床, 大约2-5 分钟。不要让鼠标无人看管, 在其他老鼠的陪伴下, 直到它完全从麻醉中恢复。</li>
</ol>2. CMAP 在后肢和前肢中的测定
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57741/57741fig1.jpg"> 图1。CMAP 测量电极的定位.电极的位置显示后 (A) 和前肢 (B)。电极编号如下: 1: 阳极和 2: 阴极刺激电极, 3: 有源记录电极, 4: 参考电极, 和 5: 接地电极。<a href="/files/ftp_upload/57741/57741fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>使用 27 G 针电极为后肢和前肢 CMAP 测量。有关电极定位的推荐位置, 请参见图 1 。</li>
	<li>将电极放置在后肢上, 如下所示。<ol>
<li>将鼠标放在加热垫上的俯卧位置。在膝部伸展后肢, 用胶带将爪子附着在工作表面上 (图 1A)。</li>
		<li>将刺激电极置于坐骨神经凹槽两侧, 距离约2厘米 (1 = 阳极和 2 = 阴极) 在电极之间。提起皮肤, 垂直插入针通过皮肤, 并推动约5毫米的针在皮肤下不刺穿底层肌肉。</li>
		<li>同样, 放置记录电极 (3) 皮下对齐腓肠肌。将参考电极 (4) 插入到跟腱的30度角上, 并在皮肤下留下2-5 毫米针。将接地电极 (5) 以类似于刺激电极的方式放置在鼠标侧面, 但该电极的位置对测量不重要。</li>
	</ol>
</li>
	<li>将电极放在前肢上, 如下所示。<ol>
<li>将鼠标放在仰卧位置的加热垫上, 使用胶带将两个前肢伸展到身体两侧 (图 1B)。</li>
		<li>将刺激电极 (1 = 阳极和 2 = 阴极) 置于前肢两侧, 与臂丛神经对齐。提起皮肤, 垂直插入针通过皮肤, 并推动约5毫米的针在皮肤下不刺穿底层肌肉。</li>
		<li>将记录电极 (3) 置于二头肌肌肌肉的顶部, 解除皮肤。将参考电极 (4) 放在3毫米深度的步行垫上, 30 度角。将接地电极 (5) 置于鼠标侧面。 注意: 在这个设置中, 电极彼此接近。防止电极接触对方, 因为这扭曲了录音。</li>
	</ol>
</li>
</ol>3. 数据采集
<ol><li>通过推动控制器单元中的经常性刺激按钮启动刺激, 并转动强度控制器旋钮来增加刺激。刺激所有轴突使用1脉冲/秒与0.1 毫秒的刺激期。从软件的下拉菜单中选择正确的频率和持续时间。</li>
	<li>要达到 supramaximal 刺激 (5-20 ma; 在脱髓鞘条件高达 60 ma), 应用增加的刺激通过转动强度控制器旋钮, 直到 CMAP 反应的振幅停止增加。从那里, 进一步增加刺激 20%, 以确保 CMAP 振幅已达到其最大的反应。通过再次推动经常性刺激按钮结束刺激。</li>
	<li>使用 "标记" 工具指示记录中的以下点: 启动刺激、启动响应、最大正峰值和最大负峰值 (图 2)。</li>
	<li>确定延迟 (ms) 作为启动刺激到响应启动的延迟 (图 2)。将响应的起始定义为振幅开始增加的最早点。利用潜伏期评估轴突中的脱髓鞘。</li>
	<li>测量振幅 (mV) 从最大负到最大正峰值 (图 2)。利用振幅的大小来关联功能轴突的数目。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57741/57741fig2.jpg"> 图2。CMAP 反应的代表形象.描述性 CMAP 响应, 指示用于计算振幅和滞后时间的点 (A)。延迟取决于从刺激到 CMAP 反应开始的延迟。从最大负到双相波最大正峰值, 测量峰值振幅。一种健康的非转基因动物 (B) 和患病动物, 具有长时间潜伏期和减少振幅 (C) 的典型记录。<a href="/files/ftp_upload/57741/57741fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="6"><li>由于电极的精确放置会影响记录的结果值, 用 supramaximal 刺激替换电极并测量同一神经三次, 以确保获得最大的响应。使用录音的平均值。</li>
</ol>

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作者没有什么可透露的。

敏感记录方法对于评估疾病进展, 特别是治疗神经元紊乱动物模型的疗效至关重要。确定 CMAPs 是一种微创的电生理技术, 它经常用于临床和实验设置, 以评估神经传导在神经肌肉和神经病理性疾病3,18。在这里, 我们描述了一个新的应用 CMAP 记录在小鼠, 以测量神经传导在臂丛神经的前肢。该方法允许对变性小鼠模型中的神经元功能进行更全面和详细的纵向评估。
针电极比环形电极稍具侵入性, 特别是在纵向研究中, 必须注意尽量减少组织损伤。方法的一个可能的缺点是导致损伤从刺穿神经或肌肉。然而, 在仔细的皮下放置电极后, 肌肉和神经的损伤和破坏是可以预防的。与使用环形电极的方法相比, 这里提出的方法不需要从身体的大部分部位刮毛。因此, 对体温的动物没有任何不适或影响。
电极的定位对于正确和一致的 CMAP 振幅和延迟记录至关重要。最好是重新定位电极, 并在每个站点执行三次测量, 以确认最大的刺激和响应是达到的。正确的录音应该产生双相曲线, 如图 2所示。为规范该方法, 无神经损伤的非转基因小鼠是建立合适的、一致的电极定位优化刺激的最佳模型。可复用针电极适用于重复使用, 如果他们定期消毒, 例如与戊二醛在动物之间20分钟, 并检查锐度。
在正常的成年小鼠中, 所记录的 CMAP 振幅在刺激坐骨神经和臂丛后通常为 80-100 mV。这比用环形电极测量的反应大得多, 因为产生 20-40 mV8、19、20结果的环形电极的皮肤产生了更高的阻抗。在 ALS 小鼠模型中, 刺激坐骨神经或臂丛神经麻痹后 CMAP 振幅降低到 10-30 mV。CMAP 振幅的大小在年轻的动物是小的, 因为 CMAP 振幅增加在发展期间21。
我们这里描述的方法在 ALS 的小鼠模型中特别有用, 其中神经支配和随后的运动缺陷发生在后肢的早期, 而不是前肢13。除神经支配外, 该方法还能检测到 CMAP 振幅的预防或减速下降的神经。症状发作年龄后肢肌肉 CMAP 振幅显著下降, 阻碍进一步疾病进展的随访;由于 CMAP 振幅在疾病的早期阶段达到极低的值, 在疾病过程中它们不会进一步减少。相比之下, 轴突丢失的进展, 在前肢臂丛神经的速度较慢, 并提出了一个更敏感的选择, 以测量疾病进展较长的时间的疾病。此外, 较不退化的前肢可以提供一个更有效的地点, 以评估旨在增强轴突功能的治疗方法。
显然, 所提出的技术为神经肌肉紊乱小鼠模型的表征提供了新的可能性。CMAP 从坐骨神经和臂丛中注射针电极的录音, 是一种快速、重现性的方法, 用于评估后肢和前肢的轴突丢失和脱髓鞘。该方法的灵敏度, 使检测轴突的缺陷, 甚至在显著的马达赤字可以记录, 从而使这些缺陷的早期量化。此外, 反复试验的可能性减少了所需动物的数量, 并详细概述了个别动物不同部位神经肌肉和神经病变疾病的进展情况。

电生理学作为诊断工具, 在神经肌肉紊乱, 如运动神经元紊乱, plexopathies, 神经病, 神经肌肉连接紊乱, 和肌病。肌萎缩侧索硬化 (ALS), 其中主要是运动神经元受影响, 轴突损伤和肌肉麻痹1反映在减少 CMAP 振幅的神经传导研究 (NCS)。在夏科-玛丽牙病 (中) 轴突变性和脱髓鞘可以估计在周围神经使用2。该技术可用于确认诊断和评估疾病进展3,4。它能从动作电位振幅5的大小和脱髓鞘的程度来推断轴突病理, 从而降低传导速度、延长远端延迟或传导阻滞。6。
CMAP 测量是评价人和小鼠神经传导的一种快速、灵敏的方法。而在患者中, 在不同的地点例行执行的 CMAP 记录不同的神经和肌肉, 在小鼠, 通常只做了坐骨神经评估神经功能的后肢。然而, 在一些动物研究中, 记录 CMAP 在前-和后肢都是有利的, 例如, 遵循 ALS 和后肢之间的差异性疾病进展的小鼠模型。
本文介绍了用针电极记录小鼠前肢的 CMAPs 的方法。此外, 我们提供了一种方法来测量 CMAPs 从后肢, 同样与针电极。对后肢与环形电极 CMAPs 的测量, 已经提出了7,8。使用针电极记录 CMAPs 是一种快速的测量方法, 它不需要剃须毛皮, 并且测量后和前肢的过程只需要10分钟每动物为一位老练的研究员。此外, 这种微创方法是可行的重复测量, 以允许纵向随访的多神经的动物。

<table><tbody><tr><td>Resuable subdermal needle electrode, Pl/Ir</td><td>Technomed</td><td>TE/S61-434</td><td>The Needle is 13 mm (0.51&quot;) in length, 0.4 mm (27G) in diameter</td></tr><tr><td>Natus electrodiagnostic system</td><td>Natus Neurology</td><td>UltraPro S100</td><td>EMG device</td></tr><tr><td>Synergy</td><td>Natus Neurology</td><td>version 20.1.0.100</td><td>EMG software for UltraPro S100</td></tr><tr><td>Physitem Controller</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td>Heating pad</td></tr><tr><td>combi-vet Base Anesthesia System Digital Flowmeter with TEC 3 Vaporize</td><td>Rothacher &amp;amp; Partner</td><td>CV 30-301-D</td><td>Isoflurane Vaporizer and flowmeter</td></tr><tr><td>Iso-Vet 1000 mg/g&amp;nbsp;</td><td>Piramal Healthcare UK Limited</td><td>AP/DRUGS/220/96</td><td>Isoflurane</td></tr><tr><td>SOD1-G93A mice</td><td>The Jackson Laboratory</td><td>#002726</td><td>ALS tg and non-tg control littermates, only females</td></tr><tr><td>PrP-hFUS-WT3 mice</td><td>The Jackson Laboratory</td><td>#017916&amp;nbsp;</td><td>ALS tg and non-tg control littermates, all groups balanced for males and females</td></tr><tr><td>C57BL/6Jax mice</td><td>The Jackson Laboratory</td><td>#000664</td><td>Non-tg mice for axotomy, male and female</td></tr><tr><td>C61-PMP22 mice</td><td>Mouse line was generously donated&amp;nbsp; by Prof. M. Sereda (The Max Planck Institute of Experimental Medicine, G&amp;ouml;ttingen, Germany).</td><td></td><td>CMT tg and non-tg control littermates, all groups balanced for males and females</td></tr></tbody></table>

in vivo electrophysiological measurement of compound muscle action potential from the forelimbs in mouse models of motor neuron degeneration

评估神经轴突的功能提供了有关神经肌肉紊乱进展的详细信息。电生理记录提供了一种敏感的方法来测量神经传导的人和啮齿动物模型。为拓宽小鼠肌电图的技术可能性, 本文介绍了用针电极测量前肢臂丛神经的复合肌动作电位 (CMAPs)。CMAP 的录音后, 刺激坐骨神经在后肢曾被描述。新引入的方法允许在额外的站点上对神经电导率进行评估, 从而提供了对神经肌肉功能的更深刻的概述。该技术提供了有关功能轴突的相对数目和髓鞘形成水平的信息。因此, 该方法可用于评估轴索疾病和脱髓鞘的条件。这种微创方法不需要提取神经, 因此它适用于对同一动物进行纵向随访的重复测量。类似的录音是在临床设置, 以强调翻译相关性的方法。

CMAPs 的电生理测量是一种微创和非常敏感的方法, 随着时间的推移神经肌肉功能。这里描述的技术允许对小鼠前肢神经传导进行评估, 从而提供对神经功能的洞察力。
CMAP 振幅和潜伏期的测量从后和前肢在疾病过程中的两个小鼠模型的 ALS, SOD1-G93A9和PrP-hFUS-WT3 10 (图 3), 并在一个小鼠模型, C61-PMP2211,12(图 4)。als 小鼠模型是通过过度表达 als 相关的人类基因, 即突变SOD1或野生型FUS。在这两种模型中, 小鼠发育肌萎缩, 类似于渐进性运动神经元变性导致瘫痪。在非转基因 littermate 控制中, 后肢和前肢的 CMAP 振幅没有随时间变化 (图 3A)。另一方面, SOD1-G93A 小鼠后肢坐骨神经的 CMAP 振幅显著下降, 甚至在60天左右症状出现前 (而第一个运动症状通常在三月的年龄被观察到)13.该年龄在非转基因 (非 tg) 窝的振幅为 90 mV, 而 SOD1-G93A 小鼠则只有 30 mv。随着疾病进展到150天的晚期症状阶段, 振幅的进一步下降幅度只有最小。与后肢坐骨神经相比, 前肢臂丛神经的 CMAP 振幅下降, 从而导致轴突退化。在前肢中, 随着 CMAP 振幅从 70 mv 降低到 30 mv, 在这些小鼠的运动缺陷显示之前和之后, 疾病进展也更加明显。
在 ALS 的 PrP-hFUS-WT3 小鼠模型中, 运动缺陷的发生大约在28天10岁时开始, 这与 CMAP 振幅下降的起始相重合。这是一个更加速的疾病模型, 因为小鼠接近结束阶段大约在65天的年龄。与前肢臂丛神经相比, 后肢坐骨神经的 CMAP 振幅下降较快, 表明后肢早期轴突变性 (图 3D)。这一观察支持这两种小鼠模型的临床观察, 因为后肢的瘫痪明显早于在疾病过程后期仍能正常工作的前肢。
一般来说, 与后肢相比, 前肢的潜伏期从刺激到启动的时间较短 (图 3B, E)。这仅仅是由于刺激和记录电极之间的距离较短。延迟提供了轴突的髓鞘形成水平的指示。我们的观察是, CMAP 潜伏期是延长在疾病进展中的小鼠模型的 als, 虽然 als 不是脱髓鞘疾病。这很可能是由于失去了更大, 更快的导电马达轴突。
C61-PMP22 小鼠 overexpressing 3-4 份人类 PMP22 和杂合子小鼠重述了一个非常温和的 CMT1A 疾病表型, 轻度脱髓鞘和减少 CMAPs, 但没有可见表型11,12。在 1.5-2 岁的 C61-PMP22 小鼠, CMAP 振幅减少, 潜伏期延长, 在后肢和前肢 (图 4)。与健康主题的记录相比, 显示的振幅和延迟响应的代表性记录分别在图 2C(B) 中列出。前肢的 CMAP 潜伏期不像后肢那样受影响。这与 CMT1A 患者是一致的, 因为更经常的患者有严重减少或无法察觉的 CMAPs 在下肢由于病理生理学性质的作为一个长度依赖性障碍14。此外, 疾病严重程度与 CMAP 振幅相关, 而不是潜伏期或传导速度, 因为振幅与轴突完整性的程度相关14,15。然而, 结果表明, 该方法对检测脱髓鞘过程 (如 CMT1A 中观察到的) 有足够的敏感性。
在非转基因组中, 振幅和潜伏期的变化率最低 (分别为变异系数2-15% 和 1-13%)。在所有转基因病例中, 测量值 (振幅8-51% 和潜伏期1-21% 的变异系数) 有更多的变化, 这很可能是由动物之间疾病进展的差异引起的。在所有情况下, 变异在后肢和前肢中都是相似的。针和表面电极的使用变化被报告为类似的16。
非转基因和 ALS 模型所需的刺激强度没有很大差异 (图 3C, F)。同样, 在这些病例中, 达到 supramaximal 刺激所需的刺激也类似于前-后肢和 5-12 mA 之间的变化。在 C61-PMP22, 增加刺激强度的要求得到了确认17 , 同样的表型在小鼠身上可见 (图 4C)。这种现象是由增加的电阻阻抗从肥厚的 endoneurial 改变17。
为了证实从前肢中记录的 CMAP 振幅是由于神经刺激而非肌肉刺激, 我们在5月大的非转基因 C57BL/6Jax 小鼠 (男性和女性) 的臂丛神经上进行了单侧部分切断 (图 5)。切断将 CMAP 振幅从 90 mv 降低到 20 mv, 表明大部分轴突在操作中断开。对侧前肢或后肢的振幅没有变化。这一结果强烈表明, 在二头肌肌中检测到的反应是由于神经刺激, 并没有导致肌肉刺激。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57741/57741fig3.jpg"> 图3。CMAP 的振幅, 潜伏期, 和必要的刺激, 在疾病的过程中的后-和前肢的 ALS 小鼠模型.SOD1-G93A (AC) 和 PrP-hFUS-WT3 (d-F) 转基因 (tg) 小鼠和非转基因 (非 tg) 窝是在出现的运动症状, 在症状阶段, 并在疾病过程的晚症状阶段, 在年龄 57, 91, 147 天 (d) 或在 29, 38, 53 天为 SOD1-G93A 和 PrP-hFUS-WT3 小鼠, 分别。黑色: 非转基因后肢, 黑色虚线: 非转基因前肢, 灰色: 转基因后肢, 灰色虚线: 转基因前肢。结果表明, 在非转基因动物的后肢和前肢中, 振幅 (A、 D) 稳定。在转基因动物中, 在疾病过程中振幅下降。潜伏期 (B, E) 受此病影响较小, 并观察到后和前肢之间的主要差异, 无论基因型。所需刺激 (C、 F) 的变化在所有群体中都是极小的。对于 SOD1-G93A N = 4 在所有组除 tg 147 d, N = 3。对于年龄组29、38和53的 PrP-hFUS-WT3 小鼠, N 为非 tg 4、5和 4, 分别为 tg 7、5和3。符号表示组之间的差异如下: *: 非 tg 后肢与 tg, #: 非 tg 前肢与 tg 前肢, ¤: 非 tg 与非 tg 前肢, 灰 *: tg 与 tg 前肢。双路方差分析与 Tukey 的多比较测试, *: p < 0.05, **: p < 0.01, **: p < 0.001, ***: p < 0.0001. #: p < 0.05, #: p < 0.01, # #: p < 0.001, # #: p < 0.0001. ¤: p < 0.05, ¤¤: p < 0.01, ¤¤¤: p < 0.001, ¤¤¤¤: p < 0.0001。<a href="/files/ftp_upload/57741/57741fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57741/57741fig4.jpg"> 图4。CMAP 振幅, 潜伏期, 以及 CMT1A 小鼠后肢和前肢所需的刺激.C61-PMP22 转基因 (tg) 小鼠和非转基因 (非 tg) 窝测定了 1.5-2 岁。在转基因小鼠的后肢和前肢中, 振幅 (A) 均降低。在小鼠的所有肢体, 潜伏期 (B) 延长, 甚至在前肢的细微变化检测与此测量。C61-PMP22 小鼠对刺激强度 (C) 的要求增加, 与 CMT1A 患者的检测表型相似。结果为非 tg n = 4 和 tg n = 3 提供了平均值。双路方差分析与 Sidak 的多比较测试, **: p < 0.01, ***: p < 0.0001。<a href="/files/ftp_upload/57741/57741fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57741/57741fig5.jpg"> 图5。前肢动作电位是由神经刺激引起的.为了排除观察到的 CMAP 反应是由肌肉刺激引起, (部分) 切断在臂丛神经上进行的可能性。CMAP 振幅 (A) 和潜伏期 (B) 记录在 (前) 和4天后 (后) 切断的臂丛神经在成年非转基因小鼠。切断减少了 CMAP 振幅, 表明反应是神经刺激引起的。黑色: 后肢, 灰色: 对侧前肢, 灰色虚线: 同侧前肢。结果显示为平均值 * SD, N = 2。<a href="/files/ftp_upload/57741/57741fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration at JoVE.com

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

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

神经传导的测量是评价变性小鼠模型的有用工具, 但它通常只用于刺激后肢的坐骨神经。在这里, 我们描述了一个技术, 以测量在体内的复合肌肉动作电位 (CMAP) 的小鼠前肢肌肉支配的臂丛神经。

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

in-vivo-electrophysiological-measurement-compound-muscle-action

Research

JoVE Journal

Neuroscience

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

19.2K 次观看.  KU Leuven - University of Leuven. 神经传导的测量是评价变性小鼠模型的有用工具, 但它通常只用于刺激后肢的坐骨神经。在这里, 我们描述了一个技术, 以测量在体内的复合肌肉动作电位 (CMAP) 的小鼠前肢肌肉支配的臂丛神经。

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

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

电电机组数估计（MUNE）在小鼠后肢肌肉测量复合肌肉动作电位（CMAP）

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

科研

行为学

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 ...

神经科学

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

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

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

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 ...

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 the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

<meta charset="utf8"></head><body>小鼠耳长提肌电穿孔和神经损伤

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

<meta charset="utf8"></head><body>作者聚焦：肌肉蛋白在神经损伤后神经肌肉接头的再生作用

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

颅提肌耳长骨骼肌的体内电穿孔和短期神经再支配相结合

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

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电电机组数估计（MUNE）在小鼠后肢肌肉测量复合肌肉动作电位（CMAP）

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

在对小鼠坐骨神经体内的电测量

测量脊髓突触前抑制小鼠背根电位记录

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测量神经肌肉接头功能

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

颅提肌耳长骨骼肌的体内电穿孔和短期神经再支配相结合

颅提肌耳长骨骼肌的体内电穿孔和短期神经再支配相结合

颅提肌耳长骨骼肌的体内电穿孔和短期神经再支配相结合