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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(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. 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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。

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

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 

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。

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 (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。

the-ex-vivo-preparation-spinal-cord-slice-for-whole-cell-patch-clamp

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

2.3K 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 

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Methods Article

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

Methods Article

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

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