The authors would like to acknowledge the Painless Research Foundation for support of the work. This work was also supported by the NIH grants R01 NS080921-01 and R21 NS079897-01A1.

伦理声明:维护和使用实验动物的所有程序符合UCSF委员会对动物研究的法规，按照美国国立卫生研究院的规定动物使用和保养（出版85的指导方针进行了 - 23日，1996年修订）。加州大学旧金山分校的机构动物护理和使用委员会批准在这项研究中所使用的协议。 1.仪器，解决方案和准备餐具<ol><li>准备人工脑脊液（学联）。 <ol><li>制备500毫升10×低阳离子溶液，和500毫升的10倍的碳酸氢盐溶液（ 见表1和2的溶液制剂）。保存在4℃，使用一个月内。 </li><li>通过将60毫升的10倍低的阳离子溶液用60ml碳酸氢钠溶液制备600毫升新鲜脑脊液中，然后添加480毫升去离子水以达到600毫升最终体积。泡泡学联与卡波（5％O 2和95％的CO 2），用于在使用前至少10分钟。 </li></ol></li><li>从玻璃毛细管拉补丁吸管。 <ol><li>将薄壁毛细玻璃成吸管拉马准备录音移液器。 注:在我们的实验室中，拉马以下设置参数来达到理想的吸管状:热（510），速度（32）。为完整DRG记录了理想吸管形状应具有低的锥角逐渐细长锥形的，这最好通过试验和错误来达到。 </li><li>确保该吸管的电阻为3-5兆欧时填充有内部溶液（见溶液制备表3）。请参考轴突指南测量吸液管电阻18的详细方法。 </li></ol></li><li> 1毫克胶原酶添加到400μl的脑脊液的，得到的13单位/ ml的最终浓度。然后填写与约20微升胶原溶液各玻璃吸管。商店在-20℃下冷冻填充移液管和三周内使用它们。 </li><li>制得200毫升冷学联（约4℃）的。为了冷却学联迅速将其放入烧杯中，用密封在-20℃下冷冻保鲜膜和地点约20分钟，直到它开始冻结。放置在冰浴中和气泡与卡波金的烧杯10分钟。泡在室温其余400毫升学联与卡波。 </li><li>而脑脊液被冷却，切一次性塑料移液管的端部以放大的开口到一个直径为5毫米（这将被用于传输DRG）。收集以下手术器械:手术刀（＃15），梅奥直，弯剪，精2毫米尖咬骨钳，斜角肌挤压（齿）镊子，虹膜剪，弹簧剪刀和两个细镊子。倾含氧冷脑脊液成3玻璃培养皿（外径:10厘米）。 </li></ol> 2. DRG解剖<ol><li>麻醉大鼠（200-220 g）在PE钠ntobarbital（100毫克/千克，IP）并确认麻醉水平是通过测试脚蹬反射和眨眼反射足够。 </li><li>验证将大鼠麻醉后，刮胡子腰部区域和切开沿中线的皮肤和皮下组织。从分离的L1-S2级棘突用细骨膜椎旁肌。 </li><li>用弯剪从L1棘突削减到S2。使用直梅奥剪刀尽在暴露脊柱约为L1和S1横向切口，取出脊柱的这一部分整块并迅速淹没到冷学联（步骤1.5准备），2-3分钟。在排除腰病种付费的，安乐死是通过双边开胸进行，而老鼠仍然是深戊巴比妥麻醉下。 </li><li>冲洗掉从去除脊柱的血液和连接的组织，并转移到陪替氏培养皿用冷脑脊液。 ü瑟细咬骨钳去除薄层。切割沿中线硬膜与microscissors以暴露脊髓。轻轻抬起脊髓，并使用虹膜剪脊髓切断神经根在他们的切入点，然后取出脊髓。 </li><li>转移与按病种付费其余椎骨充满冷学联第二培养皿。识别L4和根据它们的相对位置，以横突和坐骨神经L5的DRG。 注:维护坐骨神经完好无损将有助于确定L4和L5 DRG（坐骨神经从L4-6脊神经起源）。 </li><li>使用杜蒙钳和剪刀诺伊斯从周围结缔组织释放病种付费。保持神经根和脊髓神经连接到病种付费。 </li><li>用移液管的病种进入第三培养皿进一步解剖。 </li><li>根据解剖显微镜，小心地取出尽可能的周围神经外膜的第ËDRG。 <ol><li>使用诺伊斯弹簧剪刀剪一开口，其中背部和腹部的根加入DRG，并从DRG分离神经前根。使用杜蒙＃5镊子钝头举行外膜，并用另一细镊子从滚神经外膜的DRG。 </li><li>继续尽可能的外膜去除附着使用细镊子病种付费。 注意:良好的解剖DRG重要的是要获得在下一步骤良好的消化。 </li></ol></li></ol> 3. DRG消化<ol><li>转移DRG到记录室中。使DRG，其中神经根起源的面孔下来肯定的一面。 注意:以这种方式，大部分神经元访问。 </li><li>使用锚来稳定DRG。灌流DRG含氧脑脊液在通过用蠕动泵，它被放置在一个表旁边的记录钻机相连的塑料管以0.5ml / min的速率。 </li><li>等待30分钟，以使细胞恢复。通过CCD摄像机评估DRG的红外下差分接口对比在40倍物镜放大倍率的质量（IR-DIC）光学元件。 注:按病种付费好通常包含很多轮，以及对比，神经元卫星细胞在其表面所包围。 </li><li>消化的DRG的表面的一个小区域。 注意:玻璃补丁移液器置于吸移管座，这是小接头允许管被附连到玻璃吸管与一个气密密封。 <ol><li>通过个别小口径橡胶管的两片连接两个1毫升注射器移液器持有人。把充满胶原酶入吸管持有者之一和一个空玻璃吸管成其他一一玻璃吸管。 </li><li>使用显微操作的移液管的位置就在上面的DRG。然后，在显微镜下放大，相互碰撞轻轻2移液器的尖端放大枪头（理想的情况下，直径为5-的开口10微米）。 </li><li>移动填充有酶移液器靠近DRG的表面，并简要通过管连接支架和通过移动柱塞约0.5ml注射器施加正压到含有吸管胶原酶。 注意:在压力下，酶的从吸管该流程将略有放大DRG神经元，其可作为标志酶的应用之间的空间。 </li><li>后10至15分钟，观察到剩下的外膜的碎片时，施加轻柔负压空吸管吸走的碎屑。 注意:在这种方式中，神经元和周围卫星细胞会清楚暴露并准备补丁记录。 </li><li>丢弃这两个吸管放入锐器容器。 </li></ol></li></ol> 4.膜片钳记录<ol><li>放置电极溶液（ 见表3）在冰上，以防止降解。装满玻璃的补丁吸管过滤细胞内溶液（注射器过滤器，孔径:0.2微米），并放置在探头吸管保持器吸管。通过降低吸移管到浴溶液中之前位移约1ml柱塞施加到吸管轻柔正压通过5毫升注射器。 注意:这可以防止吸移管从被堵塞。 </li><li>选择将模式旋钮放大器上的"V-钳"模式，然后在软件中打开"膜测试"接口。靠拢在显微镜下检查目标神经元的吸管。 注意:在完整的DRG制备，卫星细胞的薄层封装每个神经。 </li><li>使用从吸管正压遍历卫星细胞层直至神经元和卫星细胞的周围层之间空间的突然放大观察。继续前进对神经元的吸管，直到"酒窝"是在神经元观察到。 注:与比较从解除关联神经元的录音，正压需要是稍微大，这将有助于穿透卫星细胞层和增加成功的修补的机会。 </li><li>降低正压力。其次，实现了千兆欧姆密封，轻轻吸气。 注意:在此方法中，记录的成功率是至少70％。 </li><li>如前面19所描述获得全细胞记录的配置。 <ol><li>简单地说，通过一个简短而强劲吸力穿透神经细胞膜。可替代地，当施加抽吸使用放大器上的"扎普"功能。 </li><li>一旦全细胞模式被建立，通过在放大器转动电容和电阻补偿旋钮补偿全细胞电容和串联电阻（Rs）。 注:RS通常是5-20MΩ。 </li><li>摒弃细胞如果Rs最初是大于30兆欧;或Rs中的记录过程中超过20％的变化。也测量静息膜电位;放弃的小区，如果静息膜电位是超过-50毫伏。 </li></ol></li><li>关闭"膜测试"窗口。通过转动模式旋钮放大器选择"我钳位正常"模式。 </li><li>点击"开放协议"的软件，选择并加载协议用于测量rheobase。点击"记录"开始录制。测量输入电阻和rheobase通过注入分级系列在100 Pa的步骤去极化电流检查神经元兴奋性。 <ol><li>再次单击"开放协议"，并选择用于测量膜门槛的协议。一个500毫秒的去极化电流斜坡（2,000 PA / S）将被注入到神经元。 </li><li>使用数据采集和分析软件（ 例如 Clampfit）根据制造商的说明来分析所记录的痕迹。使用该软件的稳态IV RELAT的基础上计算的输入电阻值（R in）交付的超极化电流时ionship。移动光标来衡量rheobase和膜的阈值。 注意:以诱导该AP所需的电流的幅度被定义为rheobase，以及用于诱导的AP的最低电压被定义为膜的阈值。 </li></ol></li><li>记录的配体诱导电流。 <ol><li>填与特异性激动剂吸管。 注意:在当前的报告中，我们使用100μM的谷氨酸和100μMAITC诱导分别由谷氨酸受体和TRPA1受体介导的电流。 </li><li>检查吸管，以确保里面没有气泡。放置在移液管固定器的吸移管。吸管保持器连接与连接到药物分配系统的管。 </li><li>使用操纵器中的神经元的50微米移动吸移管。设置药物分配系统的压力为1磅和持续时间为1秒。切换记录模式到电压钳，和在-70mV夹紧。简要地通过药物分配系统施加压力来记录一个药物诱导的电流。 </li></ol></li></ol>

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<li>Hayar, A., Gu, C., Al-Chaer, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+method+for+patch+clamp+recording+and+calcium+imaging+of+neurons+in+the+intact+dorsal+root+ganglion+in+rats%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats.</a> J Neurosci Methods. 173, 74-82 (2008).</li>
<li>Yagi, J., Sumino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+a+hyperpolarization-activated+current+by+clonidine+in+rat+dorsal+root+ganglion+neurons%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons.</a> J Neurophysiol. 80, 1094-1104 (1998).</li>
<li>Ma, C., Donnelly, D. F., LaMotte, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+visualization+and+functional+characterization+of+primary+somatic+neurons%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">In vivo visualization and functional characterization of primary somatic neurons.</a> J Neurosci Methods. 191, 60-65 (2010).</li>
<li>Vit, J. P., Jasmin, L., Bhargava, A., Ohara, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Satellite+glial+cells+in+the+trigeminal+ganglion+as+a+determinant+of+orofacial+neuropathic+pain%5BTitle%5D)+AND+%22Neuron+Glia+Biol%22%5BJournal%5D)">Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain.</a> Neuron Glia Biol. 2, 247-257 (2006).</li>
</ol>

The authors declare that they have no competing financial interests.

我们报告准备膜片钳研究整个病种付费的方法。有用于制备理想的检体的几个关键要素。首先，与附后根解剖的DRG是重要的。在此之后，神经外膜需要避免对神经元损害，同时要小心除去。最后，以暴露的神经元和其周围卫星细胞，有必要以消化剩余的结缔组织。从这里所描述的方法制备的成年大鼠完整的DRG将保持良好的生存力为6至8小时，并且可以在该期间稳定地记录。它们可用于诊断相关组?...

感觉是一个有机体的生存和健康的基石。刺激的传输依赖于起始于从初级感觉神经元轴突的周神经末梢的感觉通路。初级感觉神经元，与三叉神经的脑核外，位于三叉神经节和背根神经节（背根节）。他们担任的感官信息1看门人。在perikarial膜，正如在中央和外围终端，DRG神经元表达的受体和离子通道，例如谷氨酸受体，TNFα的受体，瞬时受体电位阳离子通道亚家族V部件1（TRPV1），钠通道等 2 -7。在perikarial膜的膜片钳记录可以理解许多这些受体和渠道遍布神经细胞的功能变化。 膜片钳记录技术是STU一个强大的工具垂死的通道或受体和大量的研究活动已被应用在背根神经节8-10这项技术进行的。在大多数研究中，DRG通过切割背侧支根和脊神经靠近神经节除去。切碎后，将神经节然后被放置在导致DRG神经元，然后可记录之前立即或培养记录数天的分解酶。不幸的是，DRG神经元的解离涉及接近胞体的必要干切断。一旦分离和金黄地鼠，DRG神经元发生在膜兴奋11,12的表型变化以及变化。通常围绕它们单个神经元的胞体和卫星细胞之间的接触的损失是可能有助于这些变化13。神经元和卫星细胞之间的串扰是生理条件缺一不可，并在适应patholog的iCal条件如那些导致顽固性疼痛14,15。这将是具有挑战性的，研究使用解离的DRG制备神经元和卫星细胞之间的相互作用。 完好的DRG，另一方面，提供接近体内条件。在过去的几年里，我们的实验室，以及一些其他基团，一直使用从成年大鼠完整的DRG以调查在慢性疼痛3-5,11,15-17相关联的不同条件的初级感觉神经元的变化。虽然在这些研究中所使用的技术是有些建立，步骤一步描述尚未公布。在本手稿中，我们描述了一个方便，快捷的方式准备完整的按病种付费和他们的膜片钳记录使用。

<table><tbody><tr><td>Pentobarbital sodium</td><td>vortech Pharmaceuticals</td><td></td><td></td></tr><tr><td>syringe</td><td>BD</td><td>309659</td><td>1 ml, 5 ml.</td></tr><tr><td>scalpel</td><td>BD</td><td></td><td>size: 15</td></tr><tr><td>Mayo straight scissor</td><td>Fine Science Tools</td><td>14010-15</td><td></td></tr><tr><td>Mayo curved scissor</td><td>Fine Science Tools</td><td>14011-15</td><td></td></tr><tr><td>Rongeur</td><td>Fine Science Tools</td><td>16021-14</td><td></td></tr><tr><td>Adson toothed forceps</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Iris Scissor</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Noyes spring scissor</td><td>Fine Science Tools</td><td>15124-12</td><td></td></tr><tr><td>Bone scissors</td><td>Fine Science Tools</td><td>16044-10</td><td>Special for cutting the bones.&amp;nbsp;</td></tr><tr><td>Forceps: Dumont, Dumoxel Biologie #5</td><td>Fine Science Tools</td><td>11252-30</td><td>These have the fine tips that do not need sharpening when first purchased.</td></tr><tr><td>periosteal elevator</td><td>Sklar</td><td>97-0530</td><td></td></tr><tr><td>Dissection microscope</td><td>WILD</td><td></td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher brand</td><td>13-711-5AM</td><td></td></tr><tr><td>Petri dish (10 cm)</td><td>Pyrex</td><td></td><td>Glass petri dish can avoid damaging the tips of fine forceps</td></tr><tr><td>Collagenase (Liberase TM)</td><td>Roche</td><td>05-401-119-001</td><td>dissolve at the concentration of 13 U/ml, aliquot into glass pipette. Avoid repeated freeze and thaw.</td></tr><tr><td>filter</td><td>Thermo scientific</td><td>7232520</td><td>Filter the internal solutions for patch clamp recording to avoid clog.</td></tr><tr><td>Glass pipette</td><td>Sutter</td><td>BF150-110-7.5</td><td></td></tr><tr><td>Anchor</td><td>Havard apparatus</td><td>64-0250</td><td>stabilize the DRG to avoid drift.</td></tr><tr><td>Peristaltic pump</td><td>WPI</td><td></td><td></td></tr><tr><td>Pipette puller</td><td>Sutter</td><td>P97</td><td></td></tr><tr><td>Amplifier</td><td>Molecular devices</td><td>Axopatch 200B</td><td></td></tr><tr><td>Digitizer</td><td>Molecular devices</td><td>1440D</td><td></td></tr><tr><td>Microscope</td><td>NIKON</td><td>FN600</td><td></td></tr><tr><td>Micro-manipulator</td><td>Sutter</td><td>MPC200</td><td></td></tr><tr><td>microinjection dispense system</td><td>General Valve</td><td>Picrospitzer II</td><td>fast drug application system</td></tr><tr><td>Carbogen (95% O2, 5% CO2)</td><td>Local Medical Gas supplier</td><td></td><td></td></tr></tbody></table>

patch clamp recordings on intact dorsal root ganglia from adult rats

从背根神经节膜片钳研究（病种付费）神经元增加了我们周围神经系统的理解。目前，大部分的录音是在解离的DRG神经元，这是大多数实验室标准制剂进行。神经元特性，然而，可以通过从在获取解离的神经元使用的酶消化所得轴索损伤改变。另外，解离的神经元制剂不能完全代表DRG的微环境，因为与环绕初级感觉神经元卫星细胞接触的损失是这种方法的不可避免的结果。为了克服在使用传统的分离DRG神经元膜片钳记录，本报告中的局限性，我们描述编写完整的按病种付费和个人的初级感觉神经元在体外进行膜片钳记录的方法。这种方法可以完整的DRG的快速和简单的准备， 在模仿体内通过保持与周围卫星细胞和基底膜相关的背根神经节的条件。此外，该方法从操作避免性轴索损伤和酶消化这种游离病种付费时。此体外制剂可以附加地用于研究初级感觉神经元和卫星细胞之间的相互作用。

图1显示了制备完整的DRG补丁记录的过程。 图1A示出椎板切除。 图1b后神经节的曝光和位置表示的L3，L4和L5的DRG以除去脊髓后附的神经根。然后L4和5的DRG仔细解剖并从椎骨释放。接着，将神经外膜，围绕DRG的透明膜被去除（黄色箭头， 图1D）。到神经外膜分离的最佳位置是通过那里的背侧和腹侧根部加入在DRG的部位，由<st...

Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

膜片钳记录的成年大鼠完整背根神经节

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

patch-clamp-recordings-on-intact-dorsal-root-ganglia-from-adult-rats

Research

JoVE Journal

Biochemistry

14.3K 次观看.  University of California, San Francisco. This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

视频: Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

科研

神经科学

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

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

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

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

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

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

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

了解运动神经元对脊髓刺激的反应

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

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.

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 

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.

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 ...

<meta charset="utf8"></head><body>心肺感觉神经元胸段 T1 DRG 的钙成像

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.

<meta charset="utf8"></head><body>用于研究周围神经刺激的体内胸背根神经节 （DRG） 钙成像和心电图记录

This study presents a surgical manipulation to expose the T-DRG in anesthetized mice for in vivo calcium imaging, along with synchronous ECG recordings. This method represents a cutting-edge tool for studying peripheral electric nerve stimulation and thoracic visceral organ inputs, as well as their interactions at the primary sensory level.

用于研究周围神经刺激的体内胸背根神经节 （DRG） 钙成像和心电图记录

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. ...

膜片钳记录的成年大鼠完整背根神经节

摘要

探索更多视频

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

测量脊髓突触前抑制小鼠背根电位记录

斜脊髓切片用于神经前根刺激的制备

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

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

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

用于研究周围神经刺激的体内胸背根神经节（DRG）钙成像和心电图记录

膜片钳记录的成年大鼠完整背根神经节

摘要

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在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

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