本研究由国家自然科学基金青年基金（52207254和82301657）和中国博士后科学基金（2022M711833）资助。

机构动物护理和使用委员会批准了所有动物实验，研究是按照相关的动物福利法规进行的。
1. 动物准备
<ol><li>动物<ol><li>饲养信息：在特定的无病原体环境中饲养雄性Sprague-Dawley大鼠（出生后10-14天，P10-P14）。 注意：室温保持在20°C±2°C，湿度：50%-60%，光/暗循环12小时。动物可以自由获得食物和水。</li>
		<li>逆行标记运动神经元：将氟金（FG）注射到双侧胫骨前肌和腓肠肌中（在无菌盐水中2%，每块肌肉50μL）以在处死前2天逆行标记运动神经元。</li>
	</ol></li>
	<li>解决 方案<ol><li>制备切割溶液：混合 120 mM 氯化胆碱、2.6 mM KCl、26 mM NaHCO 3、1.25 mM NaH 2 PO4、0.5 mM CaCl 2、7 mM MgCl2、1.3 mM 抗坏血酸、15 mM 葡萄糖。在解剖和切片之前，用95%O 2和5%CO2（用KOH调节至pH 7.4）预起泡溶液30分钟。用碎冰冷却溶液。</li>
		<li>制备人工脑脊液（ACSF）：混合126mM NaCl，3mM KCl，1.2mM NaH2PO 4;1.3 mM MgCl 2、2.4 mM CaCl2、26 mM NaHCO3 和 10 mM 葡萄糖。在孵育前用95%O 2和5%CO2预起泡溶液30分钟。</li>
		<li>制备细胞内溶液：混合 126 mM K-葡萄糖酸盐、2 mM KCl、2 mM MgCl 2、0.2 mM EGTA、10 mM HEPES、4 mM Na 2 ATP、0.4 mM Na2GTP、10 mM K-磷酸肌酸和 0.5% 神经生物素（pH 7.25 和 305 mOsm/kg）。用碎冰冷却溶液。</li>
		<li>制备低熔点琼脂糖凝胶：将4 g琼脂糖溶于100 mL切割液中，用磁力搅拌转子使其充分溶解。在包埋前30分钟，将低熔点琼脂糖在微波炉中以高功率加热1分钟，然后将其转移到39°C水浴中以保持液态。</li>
	</ol></li>
	<li>仪器准备<ol><li>在灌注前10分钟将碎冰放在灌注盘上（补充图1A）。提前将解剖托盘（补充图1B）和切割托盘（补充图1C）与水带一起在-80°C下过夜。</li>
		<li>提前将带有尼龙网的培养室置于45°C的烘箱中过夜。</li>
		<li>使用低熔点琼脂糖预制35°斜坡和2毫米厚的平台（补充图1D）。凝胶凝固后，将它们放在 35 毫米培养皿的中心，以在下一个即将到来的手术中支撑脊髓。</li>
	</ol></li>
	<li>心内灌注<ol><li>通过腹膜内注射用2.5%三溴乙醇（160μL/ 10g）麻醉大鼠。通过验证对外部刺激（例如轻轻捏脚趾）缺乏反应，确保大鼠完全麻醉。</li>
		<li>确认适当的麻醉后，将大鼠仰卧并将它们固定在装有硅胶的培养皿中。</li>
		<li>在剑突尾部切开一个 5 毫米的纵向皮肤切口，然后完全扩大皮下空间。沿腹侧中线切开一个2厘米的纵向皮肤切口，充分暴露外胸壁，从上述切口开始，到胸部顶部结束。</li>
		<li>用齿形镊子提起剑突 （补充图2A），然后用细剪刀剪断隔膜。沿剑突两侧切开胸骨，打开胸部并暴露心脏（补充图2B）。 注意： 注意保留两侧的胸内血管;否则，可能会导致大量出血。</li>
		<li>使用无齿镊子提升左心室。沿左心室纵轴将一根 22 G 针插入左心室顶点（补充图 2C）。同时，观察灌注管内有节律的血液搏动，否则针头可能刺入右心室，可能导致灌注效果不佳。 注意： 不应使用齿形镊子;否则可能会导致镊子固定部位渗出额外的血液。</li>
		<li>使用细剪刀剪开右心房（补充图2C），然后在1分钟内以约2mL / s的速度手动注入100mL冰冷的灌注液。 注意：当大鼠的肝脏和爪子变得苍白，并且没有血液从右心房流出时，可以达到良好的灌注。</li>
	</ol></li>
	<li>脊髓夹层（图1）<ol><li>将大鼠置于俯卧位，分别在髂前上棘（约L4椎体水平）和胸柱曲率移动点（约T6椎体水平）处切割脊柱（图1A）。然后，立即将离体脊柱置于含氧冰冷灌注液中，洗去残留的血液和脂肪组织;该程序有利于在后续程序中保持手术区域清洁。 注意：应保留脊柱旁肌肉，有利于后续使用昆虫针固定脊柱。</li>
		<li>立即将分离的脊柱转移到解剖托盘（图1B），背侧朝上，喙端靠近操作员。用50mL连续氧化的冰冷切割溶液填充解剖托盘（图1B）。</li>
		<li>使用四个昆虫针通过穿透脊柱旁肌肉来固定脊柱（图1B）。</li>
		<li>在解剖显微镜下，用微型剪刀从喙端切开两侧的椎弓根，这可以称为“椎板切除术”（图1C）。注意不要损伤脊髓。同时，使用微齿镊子将切开的椎体抬起。</li>
		<li>椎板切除术后，不要立即将脊髓与椎管分离。取而代之的是，使用微型剪刀沿背中线切割硬脑膜，这有利于细胞和含氧ACSF之间的营养吸收（图1D）。 注意： 切勿撕裂硬脑膜。只允许用微型剪刀切割硬脑膜;否则，神经根和脊髓实质将受到严重损害！</li>
		<li>抬起脊髓的喙部，然后小心地切割约1毫米的神经根（图1E）。将脊髓与椎管分离后，使用2个昆虫针将脊髓腹侧朝上固定（图1F）。</li>
		<li>使用微型剪刀沿腹侧中线切割硬脑膜（图1F）。切断多余的神经根，保留约1毫米。 注意：神经比脊髓坚韧得多。如果保留的神经根太长（>1毫米），振动切片机不能切断神经根，这可能导致脊髓实质严重撕裂。</li>
		<li>使用微型剪刀将腰椎扩大分离到6-7毫米的长度（图1G）。</li>
	</ol></li>
	<li>包埋在低熔点琼脂糖中<ol><li>将腰椎扩大放在35°斜坡上（图1H），背侧朝上，尾端朝下。使用吸收性滤纸除去组织表面的大量水分（图1H）。</li>
		<li>将熔融的琼脂糖凝胶缓慢倒入含有腰椎扩大的培养皿中（图1I）。 注意： 不要倒得太快，否则气泡会积聚在凝胶中。</li>
		<li>将上述培养皿置于冰水混合物中，尽快冷却凝胶，有利于维持细胞的活性。</li>
		<li>将凝胶修剪成15mm x 10 mm x 10 mm立方体，并用强力胶将其安装在标本盘上（图1J）。</li>
	</ol></li>
	<li>切片<ol><li>将试样盘放入预冷冻的切割托盘中，然后倒入冰冷的切割溶液（图1K）。将 95% CO 2 和 5% O2 连续鼓泡到切割托盘中。</li>
		<li>设置振动切片机参数：厚度：350μm;速度：0.14-0.16 mm/s，振幅：1.0 mm，振动频率：85 Hz。</li>
		<li>每只动物收获 2-3 片合适的切片。每片记录 1-2 个健康的 FG+ 运动神经元，每只动物的范围为 5-6 个细胞。</li>
	</ol></li>
	<li>孵化<ol><li>使用盖玻片镊子夹住切片（图1L）并将其放入充满连续氧化ACSF的孵育室中。将孵育室置于32°C的水浴中30分钟，然后在记录前继续在室温（RT）下孵育30分钟。 注意：上述程序，从麻醉到获得第一层，应在20-30分钟内完成，以尽可能保留细胞的活力。每个切片中的运动神经元可以维持其活力约6-7小时。</li>
	</ol></li>
</ol>2. 使用SCS进行膜片钳记录（图2）
<ol><li>准备<ol><li>以约1-2 mL / min的速率用连续起泡的ACSF灌注记录室。通过蠕动泵上的控制面板单独调节流量。</li>
		<li>将切片放入录音室。使用带有尼龙线的 U 形铂丝将切片牢固地固定到位。</li>
		<li>使用红外微分干涉对比显微镜（IR-DIC）观察切片。在4倍物镜下，确认背根的长度约为1mm。找到背根进入实质的区域，然后将中央视野移动到该区域。</li>
		<li>将脉冲发生器与定制的电极连接（图2A）。</li>
	</ol></li>
	<li>SCS 配置<ol><li>通过显微操作系统将SCS的阳极放置在背中线附近（图2B）。</li>
		<li>通过显微操作系统将SCS的阴极放置在背根进入区（DREZ）附近（图2B）。</li>
	</ol></li>
	<li>细胞靶向和成像<ol><li>使用带有 10 倍物镜的 IR-DIC 大致找到运动柱的背外侧区域，大多数运动神经元都位于该区域。然后，将中央视野移动到该区域。</li>
		<li>切换到 60 倍物镜以找到具有光滑明亮表面和不可见核的健康神经元（图 2C、F）。</li>
		<li>稍微调低红外强度并打开荧光光源。将滤光片切换到宽带紫外激发滤光片（图2D，G），以选择合适的FG阳性（FG +）运动神经元（图2E，H）。</li>
		<li>使用抽吸电极对背根施加 1x 运动阈值刺激。如果在运动神经元中检测到诱发电位（补充图3），则确认背根的活动完好无损。如果没有，则应丢弃此切片。</li>
	</ol></li>
	<li>膜片钳记录<ol><li>用细胞内溶液填充微量移液器并将其插入电极支架。使用显微操作系统将移液器降低到ACSF浴中。移液器电阻范围为 5-8 MΩ。</li>
		<li>对移液器施加少量正压以吹走灰尘和细胞碎片。<ol><li>降低电极以接近电池。当移液器接触FG+神经元的表面时，在尖端水平处可以看到膜的小凹痕。释放正压。</li>
			<li>然后，使用注射器对移液器施加少量负压。这会产生少量的吸力，使细胞膜与玻璃移液器接触。始终注意软件界面上的总电阻，直到电阻值增加到千兆欧 （>1 GΩ）。然后，形成gigaseal。</li>
		</ol></li>
		<li>将膜电位夹在 -70 mV，然后按下放大器软件界面上的快速电容补偿按钮。轻轻施加瞬时负压使细胞膜破裂，然后按下软件界面上的 慢速电容补偿 按钮 amp扩音器。此时，获得了良好的全细胞配置。</li>
		<li>通过单击软件界面上的 IC 按钮切换到电流钳位模式，并记录静息膜电位 （RMP）。</li>
		<li>以 1-10 mA 的幅度施加 SCS 1-2 s，同时脉冲宽度和频率分别固定在 210 μs 和 40 Hz。通过缓慢增加刺激幅度来确定运动阈值，直到观察到第一个AP。</li>
		<li>在电流钳模式10,11,12 中使用流变基周围的 5 秒去极化电流注入来区分延迟和立即放电的运动神经元。即时放电运动神经元：低流变基可以诱导即时和重复放电，放电频率稳定;延迟放电运动神经元：高流变碱基可以诱导延迟发病，以加速放电速率重复放电（图3）。</li>
		<li>当 SCS 关闭时，继续记录用于捕获自发 AP 发射的膜电位。</li>
		<li>执行电压钳记录 当 SCS 打开和关闭时，兴奋性突触后电流 （EPSC） 的电压钳记录。刺激参数为 1x 运动阈值，210 μs，2 Hz。</li>
	</ol></li>
</ol>

了解运动神经元对脊髓刺激的反应

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+multimodal+neural+transmission+suggests+that+adult+spinal+networks+maintain+an+intrinsic+state+of+readiness+to+execute+sensorimotor+behaviors.">Spontaneous multimodal neural transmission suggests that adult spinal networks maintain an intrinsic state of readiness to execute sensorimotor behaviors.</a> Journal Of Neuroscience. 41 (38), 7978-7990 (2021).</li><li>Manuel, M., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intracellular+recording+of+a+lumbar+motoneuron+and+the+force+produced+by+its+motor+unit+in+the+adult+mouse+in+vivo.">Simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo.</a> Journal of Visualized Experiments: JoVE. (70), e4312 (2012).</li><li>Luo, X., Wang, S., Rutkove, S. B., Sanchez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhomogeneous+volume+conduction+effects+affecting+needle+electromyography:+an+analytical+and+simulation+study.">Nonhomogeneous volume conduction effects affecting needle electromyography: an analytical and simulation study.</a> Physiological Measurement. 42 (11), (2021).</li><li>Barra, B., 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=Epidural+electrical+stimulation+of+the+cervical+dorsal+roots+restores+voluntary+upper+limb+control+in+paralyzed+monkeys.">Epidural electrical stimulation of the cervical dorsal roots restores voluntary upper limb control in paralyzed monkeys.</a> Nature Neuroscience. 25 (7), 924-934 (2022).</li><li>Powell, M. P., 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=Epidural+stimulation+of+the+cervical+spinal+cord+for+post-stroke+upper-limb+paresis.">Epidural stimulation of the cervical spinal cord for post-stroke upper-limb paresis.</a> Nature Medicine. 29 (3), 689-699 (2023).</li><li>Wenger, N., 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=Spatiotemporal+neuromodulation+therapies+engaging+muscle+synergies+improve+motor+control+after+spinal+cord+injury.">Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.</a> Nature Medicine. 22 (2), 138-145 (2016).</li><li>&#214;zyurt, M. G., Ojeda-Alonso, J., Beato, M., Nascimento, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+longitudinal+lumbar+spinal+cord+preparations+to+study+sensory+and+recurrent+motor+microcircuits+of+juvenile+mice.">In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice.</a> Journal of Neurophysiology. 128 (3), 711-726 (2022).</li><li>Moraud, E. M., 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=Mechanisms+underlying+the+neuromodulation+of+spinal+circuits+for+correcting+gait+and+balance+deficits+after+spinal+cord+injury.">Mechanisms underlying the neuromodulation of spinal circuits for correcting gait and balance deficits after spinal cord injury.</a> Neuron. 89 (4), 814-828 (2016).</li><li>Capogrosso, M., 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=A+computational+model+for+epidural+electrical+stimulation+of+spinal+sensorimotor+circuits.">A computational model for epidural electrical stimulation of spinal sensorimotor circuits.</a> Journal of Neuroscience. 33 (49), 19326-19340 (2013).</li></ol>

SCS调制的运动信息最终收敛到运动神经元。因此，以运动神经元为研究靶点可以简化研究设计，更直接地揭示SCS的神经调控机制。为了同时记录不同的刺激特征和细胞反应，膜片钳是在单细胞尺度上研究电生理特征的好方法。然而，仍然存在一些困难，包括如何保持细胞活力，如何快速将脊髓与骨结构分离，以及如何利用SCS成功诱导APs。因此，本研究旨在帮助研究人员快速掌握基本的操作技能，?...

脊髓刺激（SCS）可有效恢复脊髓损伤（SCI）后的运动功能。Andreas Rowald 等人报告说，SCS 在一天内实现了下肢运动和躯干功能1.探索SCS对运动恢复的生物学机制是制定更精确的SCS策略的关键和趋势研究领域。例如，Grégoire Courtine的团队证明，脊髓中的兴奋性Vsx2中间神经元和Hoxa10神经元是响应SCS的关键神经元，细胞特异性神经调控在SCI2后恢复大鼠行走能力是可行的。然而，很少有研究关注单细胞尺度上SCS的电机制。尽管众所周知，超阈值直流电刺激可以引发经典鱿鱼实验3,4,5中的动作电位（AP），但脉冲交变电刺激（如SCS）如何影响运动信号的产生仍不清楚。
鉴于脊髓内神经回路的复杂性，适当选择细胞群对于研究SCS的电机制非常重要。尽管 SCS 通过激活本体感受通路6 来恢复运动功能，但运动神经元是执行运动命令的最终单元，该命令来自整合本体感受信息传入输入7。因此，直接用SCS研究运动神经元的电特性可以帮助我们理解脊髓运动调节的底层逻辑。
众所周知，膜片钳是细胞电生理记录的金标准方法，具有极高的时空分辨率8。因此，本研究描述了一种使用膜片钳研究运动神经元对SCS的电反应的方法。与脑膜片钳9相比，脊髓膜片钳的难度更大，原因如下：（1）脊髓由体积小的椎管保护，需要非常精细的显微操作和严格的冰冷维护才能获得更好的细胞活力。（2）由于脊髓太细，无法固定在切割盘上，应将其浸入低熔点琼脂糖中，固化后进行修整。
因此，该方法在解剖脊髓和保持细胞活力的同时提供了技术细节，从而顺利地研究SCS对运动神经元的电机制，避免不必要的试验和错误。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>Sigma</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide tweezers</td>
			<td>VETUS</td>
			<td>36A-SA</td>
			<td>Clip a slice</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>RWD Life Science</td>
			<td>S12006-10</td>
			<td>Cut the diaphragm</td>
		</tr>
		<tr>
			<td>Fluorescence Light Source</td>
			<td>Olympus&#160;</td>
			<td>U-HGLGPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-Gold</td>
			<td>Fluorochrome</td>
			<td>Fluorochrome</td>
			<td>Label the motor neuron</td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared CCD camera</td>
			<td>Dage-MTI</td>
			<td>IR-1000E</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator&#160;</td>
			<td>Sutter Instrument&#160;</td>
			<td>MP-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter instrument</td>
			<td>P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors&#160;</td>
			<td>Jinzhong</td>
			<td>wa1020</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>Microscope for anatomy</td>
			<td>Olympus&#160;</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-toothed tweezers</td>
			<td>RWD Life Science</td>
			<td>F11008-09</td>
			<td>Lift the cut vertebral body</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>V900182</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>LUMPLFLN60XW</td>
			<td>working distance 2 mm&#160;</td>
		</tr>
		<tr>
			<td>Osmometer&#160;</td>
			<td>Advanced&#160;</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier&#160;</td>
			<td>Axon&#160;</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp digitizer</td>
			<td>Axon&#160;</td>
			<td>Digidata 1550B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter&#160;</td>
			<td>Mettler Toledo&#160;</td>
			<td>FE28</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Anchor</td>
			<td>Multichannel system</td>
			<td>SHD-27H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinal cord stimulatior</td>
			<td>PINS</td>
			<td>T901</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezer</td>
			<td>RWD Life Science</td>
			<td>F13030-10</td>
			<td>Lift the xiphoid</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide band ultraviolet excitation filter</td>
			<td>Olympus&#160;</td>
			<td>U-MF2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo preparation of spinal cord slice for the whole-cell patch-clamp recording in motor neurons during spinal cord stimulation

脊髓刺激（SCS）可有效恢复脊髓损伤（SCI）后的运动功能。由于运动神经元是执行感觉运动行为的最终单位，因此直接研究SCS运动神经元的电反应可以帮助我们理解脊髓运动调节的底层逻辑。为了同时记录不同的刺激特征和细胞反应，膜片钳是在单细胞尺度上研究电生理特征的好方法。然而，实现这一目标仍然存在一些复杂的困难，包括维持细胞活力、快速将脊髓与骨结构分离，以及利用SCS成功诱导动作电位。在这里，我们提出了一个详细的方案，使用膜片钳以高时空分辨率研究运动神经元对SCS的电反应，这可以帮助研究人员提高分离脊髓和维持细胞活力的技能，以顺利研究SCS对运动神经元的电机制，避免不必要的试验和错误。

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Andreas Rowald et al. reported that SCS enables lower limb locomotor and trunk function within a single day1. Exploring the biological mechanism of SCS for locomotor recovery is a critical and trending research field for developing a more precise SCS strategy. For example, Gr&#233;goire Courtine&#39;s team demonstrated that excitatory Vsx2 interneuron and Hoxa10 neurons in the spinal cord are the key neurons to response to SCS, and cell-specific neuromodulation is feasible to restore the rat walking ability after SCI2. However, few studies focus on the electrical mechanism of SCS at a single-cell scale. Although it is well-known that the suprathreshold direct current stimulus can elicit the action potentials (APs) in the classic squid experiment3,4,5, how the pulsed alternating electrical stimulation, such as SCS, affects the motor signal generation is still unclear.
Given the complexity of intraspinal neural circuits, appropriate selection for cell population is important for investigating the electrical mechanism of SCS. Although SCS restores motor function by activating the proprioceptive pathway6, the motor neurons are the final unit to execute the motor command, derived from integrating proprioception information afferent input7. Therefore, directly studying the electrical characteristics of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation.
As we know, patch-clamp is the golden-standard method for cellularly electrophysiological recording with extremely high spatiotemporal resolution8. Therefore, this study describes a method using a patch clamp to study the electrical responses of motor neurons to SCS. Compared with the brain patch-clamp9, the spinal cord patch-clamp is more difficult due to the following reasons: (1) The spinal cord is protected by the vertebral canal with tiny volume, which requires very fine micromanipulation and rigorous ice-cold maintenance to obtain better cell viability. (2) Because the spinal cord is too slender to be secured on the cutting tray, it should be immersed in low-melting point agarose and trimmed after solidification.
Hence, this method provides technical details in dissecting the spinal cord and maintaining the cell viability at the same time so as to smoothly study the electrical mechanism of SCS on motor neurons and avoid unnecessary trials and mistakes.

作者聚焦：推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

Spinal cord stimulation can help patients resolve most motion functions after injury. Most neurons are to execute sensory mode behaviors. Studying the electric response of most neurons can aid in understanding the underlying logic of spinal load neuromodulation.A patch clamp is the golden standard method for cellular electrophysiological recordings with the extremely high spatial temporal resolution. Therefore, this study describes a method using a patch clamp to simultaneously record the aortas stimulus characteristics and cellular responses of motor neurons at a single-cell scale. Compared with the brain patch clamp, the spinal cord patch clamp is more difficult, because the spinal cord is well protected by the vertebral canal with tiny volume.Therefore, this protocol provides technical details in quickly micro dissecting the spinal cord and reverse maintenance to obtain better cell viability. Patch clamp allows a unique understanding of the synaptic transmissions and the understanding of action potentials, coding pattern activities by the spinal cord stimulation.

由于精细操作期间严格的低温维护（补充图1、补充图2和图1），细胞活力足以进行后续的电生理记录。为了尽可能地模拟临床场景，我们使用显微操作将 SCS 阴极和阳极分别放置在背中线和 DREZ 附近（图 2），这可以启动背角中的神经信号传播到运动柱背外侧区域的运动神经元。在这项研究中，我们使用FG定位直径为20-50μm的运动神经元。<st...

Watch this Scientific Journal Video about Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology at JoVE.com

该协议描述了一种使用膜片钳研究运动神经元对脊髓刺激（SCS）的电反应的方法，具有高时空分辨率，这可以帮助研究人员提高分离脊髓和同时维持细胞活力的技能。

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

脊髓刺激可以帮助患者在受伤后解决大多数运动功能。大多数神经元执行感觉模式行为。研究大多数神经元的电反应可以帮助理解脊髓负荷神经调控的潜在逻辑。膜片钳是细胞电生理记录的黄金标准方法，具有极高的空间时间分辨率。因此，本研究描述了一种使用膜片钳在单细胞尺度上同时记录运动神经元的主动脉刺激特征和细胞反应的方法。与脑膜片钳相比，脊髓膜片钳的难度更大，因为脊髓被体积小的椎管很好地保护了。因此，该协议提供了快速显微解剖脊髓和反向维持以获得更好的细胞活力的技术细节。膜片钳可以对突触传递和动作电位的理解有独特的理解，通过脊髓刺激编码模式活动。

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Research

JoVE Journal

Neuroscience

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

1.4K 次观看.  Tsinghua University. 脊髓刺激可以帮助患者在受伤后解决大多数运动功能。大多数神经元执行感觉模式行为。研究大多数神经元的电反应可以帮助理解脊髓负荷神经调控的潜在逻辑。膜片钳是细胞电生理记录的黄金标准方法，具有极高的空间时间分辨率。因此，本研究描述了一种使用膜片钳在单细胞尺度上同时记录运动神经元的主动脉刺激特征和细胞反应的方法。与脑膜片钳相比，脊髓膜?...

视频: 作者聚焦：推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

科研

神经科学

Spinal Cord Dissection for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After exposing the heart of an anesthetized rat, cut the right atrium using fine scissors and inject 100 milliliters of chilled perfusing fluid at approximately two milliliters per second within one minute. Position the rat in the prone position. Cut the spine at the anterior superior iliac spine and the curvature shifting point of the thoracic column.Immediately place the isolated spine into the oxygenated ice cold perfusing solution to wash off residual blood and fat tissue. Then transfer the isolated spine to the anatomical tray with its dorsal side up and the rostral end near the operator. Fill the tray with 50 milliliters of continuously oxygenated ice cold cutting solution.Under a dissection microscope, cut the vertebral pedicles on both sides from the rostral end using micro scissors. Next, delicately sever the dura mater along its dorsal midline with micro scissors. Lift the rostral part of the spinal cord carefully and cut the nerve root, leaving about one millimeter reserved.After the spinal cord has been separated from the vertebral canal, secure it with the ventral side facing up using two insect pins. Trim any excessive nerve roots, leaving about one millimeter intact. Then separate the lumbar enlargement to a length of six to seven millimeters using a micro scissor.

脊髓解剖用于运动神经元中全细胞膜片钳记录

Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After dissecting the spinal cord and separating the lumbar enlargement, place the lumbar enlargement on a 35 degree slope with the dorsal side up and the caudal side down. Absorb any excess water on the tissue surface using an absorbent filter paper. Carefully pour the molten agarose gel into the Petri dish containing the lumbar enlargement.Position the Petri dish in the ice water mixture to ensure rapid gel cooling. Shape the gel into a cube of 15 by 10 by 10 millimeters, and mount it onto the specimen disc using super glue. Adjust the vibratome settings to a thickness of 350 micrometers, a speed of 0.14 to 0.16 millimeters per second, an amplitude of one millimeter, and a vibration frequency of 85 hertz.Now, use the cover slide tweezers to clip a slice and place it into the incubation chamber, filled with continuously oxygenated artificial cerebrospinal fluid.

用于运动神经元全细胞膜片钳记录的脊髓切片的制备

Patch-Clamp Recording to Study the Electrical Responses of Motor Neurons to Spinal Cord Stimulation

To begin, place the prepared spinal cord slice in the recording chamber. Then place the anode of the micro-manipulation system near the dorsal midline in the cathode close to the dorsal root entry zone for proper spinal cord stimulator positioning. Now switch to a 60X objective lens to identify a healthy neuron having a smooth and bright surface without visible nuclei.Slightly decrease the infrared intensity, turn on the fluorescent light source, and adjust the light filter to the wideband ultraviolet excitation option. Now, select an appropriate Fluoro-Gold-positive motor neuron and gradually lower the electrode until it is near the neuron. As the pipette contacts its surface, look for a small membrane indentation at the tip level and release the previously-applied positive pressure.Then apply negative pressure to the pipette using a syringe and wait until the resistance value rises to gigaohms on the software interface. Clamp the membrane potential at minus 70 millivolts. Press the fast capacitance compensation button on the amplifier's software interface, then apply a brief negative pressure to rupture the cell membrane and press the slow capacitance compensation button on the amplifier's software interface.Apply the spinal cord stimulation for one to two seconds keeping the amplitude between one and 10 milliamperes. Determine the motor threshold by gradually increasing the stimulation amplitude until the first action potential is observed. Distinguish delayed and immediate firing motor neurons using a five-second depolarizing current injection around rheobase in the current clamp mode.After turning off the spinal cord stimulation, continue recording the membrane potentials to capture the spontaneous action potentials firing. When the amplitude of spinal cord stimulation was raised, the membrane potential was increased, and the action potentials were triggered every 10 to 20 pulses. After the spinal cord stimulation was turned off, the neuron fired a series of spontaneous action potentials for a short period of time.Then, the resting membrane potential returned to minus 65 millivolts.