膜片钳记录的成年大鼠完整背根神经节
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Pentobarbital sodium | vortech Pharmaceuticals | ||
| syringe | BD | 309659 | 1 ml, 5 ml. |
| scalpel | BD | size: 15 | |
| Mayo straight scissor | Fine Science Tools | 14010-15 | |
| Mayo curved scissor | Fine Science Tools | 14011-15 | |
| Rongeur | Fine Science Tools | 16021-14 | |
| Adson toothed forceps | Fine Science Tools | 11027-12 | |
| Iris Scissor | Fine Science Tools | 14084-08 | |
| Noyes spring scissor | Fine Science Tools | 15124-12 | |
| Bone scissors | Fine Science Tools | 16044-10 | Special for cutting the bones. |
| Forceps: Dumont, Dumoxel Biologie #5 | Fine Science Tools | 11252-30 | These have the fine tips that do not need sharpening when first purchased. |
| periosteal elevator | Sklar | 97-0530 | |
| Dissection microscope | WILD | ||
| Transfer pipette | Fisher brand | 13-711-5AM | |
| Petri dish (10 cm) | Pyrex | Glass petri dish can avoid damaging the tips of fine forceps | |
| Collagenase (Liberase TM) | Roche | 05-401-119-001 | dissolve at the concentration of 13 u/ml, aliquot into glass pipette. Avoid repeated freeze and thaw. |
| filter | Thermo scientific | 7232520 | Filter the internal solutions for patch clamp recording to avoid clog. |
| Glass pipette | Sutter | BF150-110-7.5 | |
| Anchor | Havard apparatus | 64-0250 | stabilize the DRG to avoid drift. |
| Peristaltic pump | WPI | ||
| Pipette puller | Sutter | P97 | |
| Amplifier | Molecular devices | Axopatch 200B | |
| Digitizer | Molecular devices | 1440D | |
| Microscope | NIKON | FN600 | |
| Micro-manipulator | Sutter | MPC200 | |
| microinjection dispense system | General Valve | Picrospitzer II | fast drug application system |
| Carbogen (95% O2, 5% CO2) | Local Medical Gas supplier |
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