This work was supported by the NINDS (grant #1R01NS079702 to A.C.L.) and the SURP Program at Thomas Jefferson University (M.M.).

实验程序批准的托马斯·杰斐逊大学制度的动物护理和使用委员会，并符合欧洲共同体理事会指令（2010年/ 63 / EU，六百○九分之八十六/ EEC和87-848 / EEC），美国国立卫生研究院指南传导关心和使用实验动物，以及神经科学学会的在神经科学研究中使用动物的政策。 1.复合肌肉动作电位（的CMap） <ol><li>准备的动物： <ol><li>麻醉使用吸入剂（异氟烷）在1-2％递送到作用或通过注射（氯胺酮，甲苯噻嗪，乙酰丙嗪）大鼠。注射麻醉的用量如下：95.0毫克/千克氯胺酮，10.0毫克/千克甲苯噻嗪，0.075毫克/千克乙酰丙嗪的。 注：我们成功地同时使用吸入并进行该CMAP记录协议时，注射麻醉方法，没有偏好。 </li><li>该动物必须保持在适当的一在手术前nesthetic深度开始，通过手术结束时，与直到手术后丁丙诺啡已生效。确认正确麻醉进行了简短的脚趾捏来确定一个撤军响应未引出。此外，测试角膜反射用棉签。整个手术过程中，确定心脏速率和呼吸率没有增加，这表明麻醉深度变得太轻。 </li><li>适用于动物的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>外科医生应洗他/她的手开始术前消毒剂（洗必泰擦洗）。外科医生应戴无菌手套，口罩和礼服或干净的白大褂。所有仪器应洗净并在手术前高压灭菌。在高压灭菌仪器的水平，以验证灭菌完成之前添加的工具包内灭菌指示条。外科医生应验证钢带上的整条生产线是黑色的手术开始前。大盘在包装盒外面，指示釜磁带以及。如果手术是可以在几个动物在同一天进行的，清洁工具在手术之间和消毒，在一个玻璃珠灭菌。如果必要的话，通过将器械的手柄中的消毒器在适当的时间，用无菌止血钳除去仪器，冷却它在无菌场，然后放置的官能端消毒的玻璃珠灭菌仪器的两端仪器进入玻璃珠灭菌的时间适当的时期。 </li><li>一旦动物被麻醉，代替外科板与背表面向下和腹部朝上以便带可用于保护动物的前肢外科板。 </li><li>剃腹面尾端开始于颅底，并确保周围腹部上的中线的任一侧的区域是sh的AVED取决于哪个半膈正在被记录。适用碘伏对皮肤的剃表面。 </li></ol></li><li>对于CMAP录音准备： <ol><li>以下动物制备，地点接地电极皮下尾（ 图1）。 </li><li>将参考电极皮下对侧下腹部区域。 </li><li>将导体凝胶上自粘表面带或盘记录电极（表面条状电极的尺寸：0.5×3厘米），然后横向放置表面电极沿着单侧肋缘。 </li><li>将刺激电极经皮0.5厘米除了横向到气管和优越的锁骨。将电极通过深层皮肤大约为1.0公分。固定用手电极使它们的位置不与随后的刺激移动。 注：角度刺激电极90°相对于皮肤在insertio部位ñ。 </li></ol></li><li>复合肌肉动作电位（CMAP）录音： <ol><li>获得使用刺激/放大器其次是计算机辅助数据分析电生理记录。 </li><li>单拉刺激刺激参数应为0.5毫秒，1.0赫兹超最大脉冲。刺激的幅度应介于6.0和8.0 V（ 图2A）。 注：在一个实验中，使用刺激强度与整个动物相同的幅度。其目的是通过使用刺激的最小振幅最大化的响应幅度。要注意的是发生在刺激时的初始响应是刺激伪迹是很重要的。另外，应注意，以获得前面所述刺激伪迹，然后接着快速CMAP反应后稳定/平坦基线CMAP响应。为了得到这样的反应，可能需要重新定位的刺激和/或记录电极。 </li><li>等待刺激之间30秒，并重复10刺激在一排，以获得平均响应（ 图2B - C）。测量从基线的幅度以峰（ 图2A）。在动物的呼吸循环的阶段进行膈神经刺激时膈神经/隔膜没有被激活。该过程可以重复在同一动物的对侧半侧隔膜。 </li><li>最后CMAP记录会话后，灌注动物，如果染色NMJ将进行。每周进行反复录制CMAP在同一动物大约一次（或选择的时间间隔）。 </li><li>继生存CMAP录音，让动物恢复，直到清醒和sternally横卧在恢复上一个温暖的循环水加热垫，并持续监测。不返回已经历手术给其他动物的公司，直到完全恢复的动物。&lt;/ LI&gt; <li>一旦sternally横卧，监测动物每12小时的第48小时（每日其后一次）。如果动物出现脱水（皮肤松弛，精神萎靡），辖液（乳酸林格氏液;皮下;每次注射1-2毫升，用旋转部位;每天2次），直到手术后的重量和流体损失稳定。在急性术后期间提供动物小碟软化食物，如果有必要鼓励吃，如果他们不能达到的电线杆盖进纸器。 </li><li>施用丁丙诺啡以0.05毫克/千克手术结束前的剂量，然后在12小时的时间间隔的第一个24小时（和取决于疼痛/压力的迹象之后）经由皮下注射（现场旋转）。如果疼痛或痛苦的迹象后发现，对待动物与丁丙诺啡。标志指示性疼痛/窘迫包括缺乏活动，发声，进食不足或饮水（脱水），过度减肥，守着，EXCessive咬或抓现场切口。在确定疼痛和痛苦用其他标准包括无反应，以刺激无关，短暂或无端发声，吃力或反常呼吸，持续的红褐色鼻眼压放电，标志着竖毛。还监测卫生状况差，将建议患病动物的证据。如果动物仍然观察到72小时的治疗后要遇险（根据疼痛/应力的标准上面概述，以及接收后的镇痛协议如上所述）安乐死的动物。 </li></ol></li></ol> 2，神经肌肉隔膜接头（NMJ）分析<ol><li>动物解剖： <ol><li>施用动物注射麻醉过量（3次剂量使用以上：氯胺酮在285.0毫克/千克，甲苯噻嗪以30.0毫克/公斤，乙酰丙嗪在0.225毫克/千克;腹腔内给药）。过量服用之后，所有的动物应该有安乐死（开胸）第二方法To确保死亡。死亡，应注意心脏和呼吸骤停，并指出动物的固定和瞳孔放大确认。 </li><li>进行剖腹探查手术刀或夹层剪刀扩大切除从zyphoid过程尾端沿中线。小心用手术刀和镊子从下面的肌肉分开皮肤和结缔组织。 </li><li>而在zyphoid过程拉起，用解剖剪去除整个膜片，以确保隔膜保持附着到周围骨中。这一点很重要，因为它会防止损坏隔膜肌肉，将允许整个膜片的成功解剖，将在清洗肌肉后续染色帮助。 注意：在这一点上，动物如果需要可灌注。 </li></ol></li><li>清洗和隔膜的染色： <ol><li>拖住了整个解剖隔膜，优越的一面朝上，硅橡胶的玻璃百毫米培养皿中4％多聚甲醛（在1％的磷酸盐制成缓冲盐水 - PBS），用于使用立体显微镜20分钟。拉伸膜片，使其绷紧，并采用昆虫针，牵制周围组织，以避免钉扎隔膜肌肉本身（ 图3）。 </li><li>肌肉与＃5镊子和小剪刀只有上表面仔细清理掉结缔组织。 注：执行，除非另有说明这种染色协议缓缓摇摆在室温下涵盖了从光所有后续步骤。确保膈肌总是完全浸没液，以避免干燥。 </li><li>染色前，准备所有必要的试剂：1×PBS，2％牛血清白蛋白（BSA）/ PBS，0.1M甘氨酸在2％BSA / PBS，4％多聚甲醛（PFA）中的1×PBS和0.2％TBP制成（0.2 ％的Triton X100在2％BSA / PBS）中。一定要预冷甲醇在-20℃。 </li><li>洗3次为在1×PBS中的10分钟。 </li><li>孵育在0.1M甘氨酸（在2发％BSA / PBS）30分钟。 </li><li>孵育在RBTX（若丹明共轭α银环蛇毒素）溶液15分钟。解RBTX如下：若丹明共轭的α银环蛇毒素1：400稀释于1×PBS中。 注意：在这个协议中，若丹明被使用，但所选择的任何荧光团可用于染色。 </li><li>洗3次为在1×PBS中的10分钟。 </li><li>在-20℃的冰箱中，孵育用MeOH 5分钟。 注：将肌肉在冷冻之前不要添加甲醇，以及紧随其后的潜伏期删除解决方案。 </li><li>洗3次为在1×PBS中的10分钟。 </li><li>在0.2％磷酸三丁酯1小时块（0.2％Triton在2％BSA / PBS。制造）。 </li><li>用初级抗体溶液（在0.2％TBP）在4℃孵育过夜，同时摇动。抗体的稀释如下：SV2（1:10）和SMI-312（1：1000）。 </li><li>洗3次10分钟，用0.​​2％磷酸三丁酯。 </li><li>孵育使用1的二级抗体：100稀释FGM1（FITC-缀合的山羊抗小鼠IgG的1;而摇动1小时制成0.2％TBP）。 注：避光的协议染色的其余部分。 </li><li>洗涤3×10分钟用1×PBS。 </li><li>再清洁周围安装和盖片之前对肌肉的上（见上文）表面结缔组织。 注：建议清洗，然后染色后立即装入样品。然而，如果需要的话，样品可以被覆盖在1×PBS中，包在石蜡膜以防止蒸发，并储存在4℃下长达1周，与改变的PBS（每天）。 </li><li>在安装在玻璃载玻片，盖玻片与非hardset的Vectashield。密封盖玻片透明指甲油的边缘，以防止干燥。商店幻灯片长期平躺在硬纸板的幻灯片文件夹在-20℃。 </li></ol></li><li>分析和共聚焦成像： <ol><li>定量分析的染色形态，并安装在20X和40X放大倍率半振膜采用直立萤光显微镜。 注：由于隔膜染色，并在整个安装的方式进行分析，肌肉颇浓。因此，大多数的荧光染色是完全失焦时的静态图像使用非共聚焦荧光显微镜拍摄。出于这个原因，最好是在开展实时分析NMJ在显微镜，因为这可以让你不断地关注“向上”和“向下”，通过肌肉的正确识别每一个单独的NMJs的形态。使用这种方法，可以很容易地按照个体运动轴突的轨迹，因为它们移动通过在空间关系突触后受体簇的组织。 </li><li>通过测量肌肉的腹侧至背侧长度，除以肌成基于从特定脊髓水平（ 图4H）地形支配模式进行分析3个独立的部分。 </li><li>在肌肉和MOV的腹侧区域开始荷兰国际集团背侧，分析所有带位于表面肌纤维的脸NMJs。不分析肌肉纤维比从表面上的第一3-4纤维更深作为解释可能是困难的，由于较差的抗体渗透（RBTX穿透更深的进入肌肉由于毒素的小尺寸相比用于标记较大抗体神经元）。 </li><li>识别每个NMJ作为完整如果神经元的预终端轴突是厚的直径和神经终端的所有区域与下面的肌肉的乙酰胆碱受体（乙酰胆碱受体）完全重叠。 </li><li>识别每个NMJ为部分失神经支配，如果底层肌肉乙酰胆碱受体的任何地区是无人居住的相应神经末端。 </li><li>识别每个NMJ尽可能完全失神经如果肌肉乙酰胆碱受体有没有相应神经末端（它也是重要的是有其他NMJs与焦点的相同字段标记的神经元，这样一方面可以确保缺乏神经末端的不单纯是由于便便中的R肌肉的该区域的抗体的渗透）。 </li><li>识别每个NMJ作为乘法支配，如果有一个以上的预终端轴突神经支配的个体NMJ（这表明出现了去神经和神经支配恢复的可能尝试的一些水平在该路口）。 </li><li>每个标识作为NMJ支配清淡如果NMJ有神经末梢与乙酰胆碱受体底层之间完全重叠，但有一个薄预终端轴突（这也表明了去神经支配和企图的神经再支配一定程度）。 </li><li>进行分析对于每一个用于上述所有NMJ类别对每只动物3识别区域。从每只动物与来自其他动物的结果来自同一个实验组平均化的数据。在小鼠半膈大鼠半侧膈肌和150-200大约200-300结应该量化在每个实验组的至少3个（优选5-6或更多）的动物。 </li><li>获得高分辨率的共聚焦图像，使用d表示出版物，用共聚焦显微镜。 </li><li>得到的Z-堆叠在0.3微米的步长为20-40微米的深度，并收集上述各结下面附加的光学部分，以确保整个突触信息被包括。 </li><li>用采集软件重建的Z系列图像到最大强度的预测。 </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+conscious+airway+resistance+and+ventilation+estimation+in+neonatal+rodents+using+plethysmography+and+a+mechanical+lung.">Novel method for conscious airway resistance and ventilation estimation in neonatal rodents using plethysmography and a mechanical lung.</a> Respir Physiol Neurobiol. 201, 75-83 (2014).</li><li>Ngo, S. T., Bellingham, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+recording+of+the+compound+muscle+action+potential+for+motor+unit+number+estimation+in+mice.">Neurophysiological recording of the compound muscle action potential for motor unit number estimation in mice.</a> Neuromethods. 78, 225-235 (2013).</li><li>Nicaise, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+phrenic+motor+neurons+induces+long-term+diaphragm+deficits+following+mid-cervical+spinal+contusion+in+mice.">Degeneration of phrenic motor neurons induces long-term diaphragm deficits following mid-cervical spinal contusion in mice.</a> Journal of neurotrauma. 29, 2748-2760 (2012).</li><li>Lepore, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+glial-restricted+progenitor+transplantation+into+cervical+spinal+cord+of+the+SOD1G93A+mouse+model+of+ALS.">Human glial-restricted progenitor transplantation into cervical spinal cord of the SOD1G93A mouse model of ALS.</a> PLoS One. 6, (2011).</li><li>Lepore, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+transplantation-based+astrocyte+replacement+is+neuroprotective+in+a+model+of+motor+neuron+disease.">Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease.</a> Nature neuroscience. 11, 1294-1301 (2008).</li></ol>

我们什么都没有透露。

由于呼吸功能受到损害双方在外伤性脊髓损伤和ALS，开发针对呼吸和具体隔膜神经支配疗法是临床相关5,6。为了全面考察呼吸功能，应使用相结合的办法方法。的CMap测量膜片由外部膈神经的刺激方式的功能性神经支配的程度，但不是内源bulbospinal呼吸驱动8。此外，这些录音不允许检查在NMJ形态变化，特别是在个人的运动轴突和突触后乙酰胆碱受体的水平。通过使用所描述的染色?...

肌萎缩性侧索硬化症（ALS）是与两个上和下运动神经元和随后的肌肉麻痹的损失相关联的衰弱运动神经元疾病。经诊断，病人的生存是唯一的2-5年平均1。膈运动神经元（PhMN）损失是ALS发病机制的一个重要组成部分。患者最终死于PhMN支配膈肌，灵感2,3的主要肌肉损失。外伤性脊髓损伤（SCI）也与相关联的呼吸困难的严重问题。每年4约12,000 SCI新病例发生是由于脊髓损伤创伤。尽管相对于位置，类型和严重程度的异质性疾病，多数SCI案件涉及外伤的脊髓型颈椎病，其结果往往是虚弱和持续性呼吸道妥协。除了ALS和脊髓损伤，其他中枢神经系统（CNS）的疾病可以关联瓦特第i膈肌呼吸功能障碍5,6。 膈神经是传出运动神经支配是同侧半隔膜和源自位于同侧颈脊髓的C3-C5水平PhMN细胞体。 PhMN输出由在称为延髓腹侧呼吸组（rVRG）7的区域降序从脑干bulbospinal输入控制。所述rVRG-PhMN隔膜电路是中央的吸气呼吸，以及其他非通气隔膜行为的控制。各种外伤和神经退行性疾病，影响该电路可导致呼吸功能及患者的生活质量产生深远的下降。下行输入从rVRG，PhMN生存，膈神经的完整性和适当神经支配的肌肉隔膜接头（NMJ）PhMNs都是必要的正常功能隔膜。它采用的技术，因此重要的是，可以定量地评价本电路在体内在ALS，脊髓损伤等中枢神经系统疾病的啮齿动物模型。 与此协议中，目标是描述实验工具用于评估在两个电生理学和形态学水平膜片PhMN支配。复合肌肉动作电位（的CMap）通过刺激一个给定的运动神经的所有传出的运动神经元的轴突，然后分析所述目标肌纤维的诱发的去极化应答被记录。此技术可用于在体内麻醉大鼠和小鼠的量化半膈由PhMNs 8的官能支配。由于这样的事实，的CMap表示同时记录所有（或至少许多/大部分）的整个半膈，它也检查个别运动轴突和肌纤维在膜片NMJ的表型，以便跟踪疾病是有用的肌纤维 - 和治疗相关的形态变化，如局部和完井德失神经支配，再生出芽及神经再支配。这可以通过在隔膜的整个贴装免疫组织化学（IHC）来完成，接着个体NMJs的详细的形态学评估整个肌肉9。结合的CMap和NMJ分析提供了一个强大的方法定量研究膈神经支配的中枢神经系统和PNS疾病的啮齿动物模型。

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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Paraformaldehyde</td>
			<td style="width:273px;">Fisher</td>
			<td style="width:131px;">T353-500</td>
			<td style="width:167px;">Make 10% solution first in de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1X Phosphate Buffered Saline, pH 7.4</td>
			<td style="width:273px;">Invitrogen</td>
			<td style="width:131px;">10010049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">2% Bovine serum albumin (2% BSA)</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">A3059-100g</td>
			<td>Dissolve 2g BSA into 100mL of 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer)</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">T9284-100mL</td>
			<td>Dissolve 0.2ml/100mL 2% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.1M Glycine</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">G-7126</td>
			<td>Add 0.185g to 25mL of 2% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">&#945;-bungarotoxin</td>
			<td style="width:273px;">Invitrogen</td>
			<td style="width:131px;">T1175</td>
			<td>Concentration 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">SMI-312&#160;</td>
			<td style="width:273px;">Sternberger Monoclonals</td>
			<td style="width:131px;">SMI312</td>
			<td>Concentration 1:1,000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">SV2</td>
			<td style="width:273px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:131px;">SV2-Supernatant</td>
			<td>Concentration 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FITC goat anti-mouse IgG1</td>
			<td style="width:273px;">Roche</td>
			<td style="width:131px;">3117731001</td>
			<td>Concentration 1:100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Silicone rubber</td>
			<td style="width:273px;">Sylgard, Dow Corning</td>
			<td style="width:131px;">Part # 184</td>
			<td>Follow instructions that come with kit: can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Vectashield fluorescent mounting medium</td>
			<td style="width:273px;">Vector laboratories</td>
			<td style="width:131px;">H-1000</td>
			<td>This is not a hard-set medium. You will need to secure the cover slip with clear nail polish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Small Spring Scissors</td>
			<td style="width:273px;">Fine Science Tools</td>
			<td style="width:131px;">15002-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dissection forceps</td>
			<td style="width:273px;">Fine Science Tools</td>
			<td style="width:131px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Software for CMAP recordings</td>
			<td style="width:273px;">Scope 3.5.6; ADI</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Disk surface electrodes</td>
			<td style="width:273px;">Natus neurology</td>
			<td style="width:131px;">019-409000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Subdermal needle electrodes</td>
			<td style="width:273px;">Natus neurology</td>
			<td style="width:131px;">019-453100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Conductive gel</td>
			<td style="width:273px;">Aquasonic&#160;</td>
			<td style="width:131px;">122-73720</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:332px;">Stimulator/recording system for CMAP recordings</td>
			<td style="width:273px;">ADI Powerlab 8SP stimulator&#160;</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Amplifier for CMAP recordings</td>
			<td style="width:273px;">BioAMP</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

functional and morphological assessment of diaphragm innervation by phrenic motor neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

成年SD大鼠接受椎板切除术或者只（无恙的控制），或单侧半挫伤SCI在C4脊髓水平10-12。在5周后，手术，峰CMAP波幅记录从半膈同侧椎板/损伤部位中的SCI大鼠（ 图2C）中的显著减少相比椎板仅控制（ 图2B）。在半膈所有NMJs均在控制未患病的野生型大鼠（ 图4A中的C）完全不变。与此相反，SOD1 G93A大鼠（ALS的啮齿动物模型）显示显著病理学在半膈N...

Watch this Scientific Journal Video about Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons at JoVE.com

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

functional-morphological-assessment-diaphragm-innervation-phrenic

Research

JoVE Journal

Neuroscience

17.4K 次观看.  Sidney Kimmel Medical College at Thomas Jefferson University. Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease. 

视频: Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

科研

神经科学

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

培养胚胎高级宫颈神经群中的大鼠交感神经元进行形态学和蛋白质学分析

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

大鼠中胃肌肉单运动单元的功能隔离

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...