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
Summary
这里介绍的方法提供了从内耳前庭神经节和螺旋神经节神经元解剖、解离、培养和膜片钳记录的详细说明。
Abstract
分离和培养的内耳神经节神经元的紧凑形态允许详细表征离子通道和神经递质受体，这些离子通道和神经递质受体有助于该群体的细胞多样性。该协议概述了成功解剖，解离和短期培养内耳双极神经元的体细胞以进行膜片钳记录所需的步骤。提供了制备前庭神经节神经元的详细说明，以及铺设螺旋神经节神经元所需的必要修改。该协议包括在穿孔贴片配置中执行全细胞膜片钳记录的说明。表征超极化激活的环核苷酸门控 （HCN） 介导电流的电压钳位记录的示例结果突出了与更标准的破裂贴片配置相比，穿孔贴片记录配置的稳定性。这些方法的组合，分离体体和穿孔膜片钳记录，可用于研究需要长期、稳定记录和细胞内环境保存的细胞过程，例如通过 G 蛋白偶联受体的信号传导。
Introduction
前庭蜗神经的双极神经元将内耳的感觉毛细胞连接到脑干。它们是有关声音和头部运动的信息的主要载体;这些重要细胞的损伤会导致耳聋和平衡障碍。神经的前庭和听觉部分分别由形态和功能多样化的不同细胞类型组成 1,2。在前庭系统中，两个传入亚群以规则或不规则的间隔自发放电2.传入尖峰时间被认为反映了离子通道组成的潜在多样性 3,4。在听觉系统中，螺旋神经节神经元 （SGN） 有两个主要亚群;I 型 SGN 接触单个内毛细胞5，II 型 SGN 接触多个外毛细胞5。来自半完整和器官型培养物的体外记录表明 I 型和 II 型 SGN 的膜特性存在差异 6,7。
在这些神经元末端发现的许多离子通道和神经递质受体也存在于它们的细胞体中。因此，可以在 体外 研究孤立的前庭和螺旋神经节体的培养物，以了解离子通道和神经递质受体如何促进这些神经元的反应。分离细胞体的紧凑形态允许高质量的电记录，适用于电压门控离子通道和神经递质受体的详细表征。易于获得具有代表性的各种神经元亚型，可对细胞多样性进行高通量分析。
本文介绍了一种在出生后第9至P20天从大鼠前庭神经节上部分离和培养解离的神经节细胞体的方法。除了成功提取、解离和接种神经节细胞所需的步骤外，还提供了将这些方法扩展到螺旋神经节的建议。这些方法是各个实验室出版物中设计的方法的演变 8,9,10。本文还包括选择健康细胞进行膜片钳记录的指南。
最后，该协议概述了使用穿孔补丁配置11 进行膜片钳记录的过程。虽然穿孔贴片配置比更常见的破裂贴片配置更耗时，技术上更具挑战性，但它更适合维持细胞质环境，允许长时间稳定的记录会话。这种记录配置的好处在这里通过相对于破裂贴片记录的超极化激活阳离子电流在穿孔贴片中提高的稳定性来说明。
该协议分为五个部分。第 1-3 节描述了可以提前准备和存储的解决方案和工具。第 4 节描述了解剖和接种前庭和 SGN 的步骤，第 5 节描述了在培养一段时间后从神经元记录的步骤。在我们手中，第 4 节和第 5 节是在连续 2 天内执行的。
Protocol
此处描述的所有动物用途均已获得南加州大学机构动物护理和使用委员会的批准。该方案中的动物是从查尔斯河实验室获得的P3至P25年龄的两性Long Evans大鼠，但这些方法可以应用于其他啮齿动物品系。在所有程序中必须穿戴实验室外套和手套，并在制备溶液时戴上防溅护目镜。 1. 准备工作 注意：本节中描述的解决方案和工具可以提前制作，以…
Representative Results
通过应用一系列电压阶跃来运行电压钳位协议，揭示了各种不同电流系列的电压依赖性激活。图1A，B显示了从VGN唤起并改编自已发表的记录13的全细胞电流的代表性示例。施加去极化电压（图1B）会激活一个内向电流（按照惯例为负），该电流会非常迅速地激活和失活（图1A）。这是钠通道 14,15 的电?…
Discussion
这里介绍的方法特定于来自分离神经元的记录;以前的研究集中在半完整制剂中轴突末端的记录。与现有的终端记录技术相比，隔离记录具有出色的空间钳位和等电位行为。此外，该协议提供了对更广泛的神经元样本的访问，因为在前庭上皮的半完整记录中只能访问带有花萼的亚群。最后，孤立的记录允许使用穿孔贴片技术，该技术可以防止细胞内环境的破坏，而细胞内环境通常会因破裂贴片记录…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Jing Bing Xue 博士和 Ruth Anne Eatock 博士对这些方法的早期贡献。这项工作得到了 NIH NIDCD R03 DC012652 和 NIH NIDCD DC012653S 的支持，R01 DC0155512到 RK 和 T32 DC009975到 DB、NN 和 KR。
Materials
| Amphotericin | Sigma-Aldrich | A4888-100MG | For perforated patch recordings. |
| ATP di-sodium | Sigma-Aldrich | A7699 | Additive to internal solution |
| B27 Supplement (50x), serum free | Thermo Fisher Scientific | 17504044 | additive to culture medium, for SGN |
| Beakers (1000, 100, 10) milliliter | |||
| bench-top centrifuge | USA Scientific | 2641-0016 | |
| Bunsen burner | |||
| CaCl2 | J.T. Baker | 1311-01 | Additive to internal solution |
| Collagenase | Sigma-Aldrich | C5318 | one out of three enzyme to digest tissue |
| Coverglass, rectangular, #1 thickness, 22×40 | Warner Instruments | 64-0707 | |
| DMSO | Biotium | 90082 | |
| Dnase I,from bovine pancreas | Sigma-Aldrich | 11284932001 | one out of three enzyme to digest tissue |
| Dumont #3 Forceps (Blunt) | Fine Science Tools | 11231-30 | |
| Dumont #5 Forceps (Fine) | Fine Science Tools | 11251-10 | |
| Dumont #55 Forceps (Fine) | Fine Science Tools | 11255-20 | |
| EGTA | Sigma-Aldrich | E0396 | Additive to internal solution |
| Electrode Puller | Narashige | PC-10 | |
| Epi-illumination light source | Zeiss | CL 1500 ECO | |
| Ethanol | Decon Labs | 2716 | for cleaning head and around dissection bench |
| Filamented Borosilicate Capillaries for electrodes | Sutter Instruments | BF140-117-10 | |
| Fine-edged dissection blade | Fine Science Tools | 10010-00 | |
| Glass Pasteur Pipettes | VWR | 14673-010 | to pull trituration pipettes |
| Heat-inactivated Fetal Bovine Serum | Thermo Fisher Scientific | 16140063 | additive to culture medium |
| HEPES | Sigma-Aldrich | H3375-100G | for pH buffering all solutions in protocol |
| Hot plate / magnetic stirrers | VWR | 76549-914 | |
| Insulated bucket filled with ice | to keep all samples and solutions cool | ||
| K2SO4, Potassium Sulfate | Sigma Aldrich | P9458-250G | Additive to internal solution |
| KCl | Sigma-Aldrich | P93333 | Additive to internal solution |
| KOH (1 M) | Honeywell | 319376-500ML | To bring internal solution to desired pH. |
| Large Spring Scissors | Fine Science Tools | 14133-13 | |
| Leibovitz medium | Sigma Aldrich | L4386 | dissection and bath solutions |
| Low-profile-bath recording chamber for culture dishes | Warner Instruments | 64-0236 | |
| luer-lok syringes, 30 ml | BD | 302832 | for drawing L-15/HEPES/HEPES solution. |
| MEM + Glutamax Supplement | Fisher Scientific | 41-090-101 | base of the culture medium |
| MgCl2-Hexahydrate | Sigma-Aldrich | M1028 | Additive to internal solution |
| microFil needle for filling micropipettes – 34 gauge | World Precision Instruments | MF34G | |
| Microforge | Narashige | MF-90 | For electrode polishing. |
| N2 Supplement (100x) | Thermo Fisher Scientific | 17502-048 | additiive to culture medium, for SGN |
| NaCl | Sigma-Aldrich | S7653 | Additive to internal solution |
| NaOH (1 M) | Thomas Scientific | 319511-500ML | for titration pH |
| Osmometer | Advanced Instruments Inc. | 3320 | |
| Oxygen, Medical grade, with adequate regulator and tubing | USC Material Management | MEDOX200 (Identifier: 00015) | for dissolving into dissection and bath solutions |
| Parafilm | Bemis | PM992 | |
| Pasteur pipette bulb (3 ml) | Fisher Scientific | 03-448-25 | bulb for trituration pipettes |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | additive to prevent contamination of culture medium |
| Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 – 60 mg/kg dosing) | MWI Animal Health | 15199 | for euthanasia |
| PES membrane filters , 0.2 micrometer | Nalgene | 566-0020 | for filtering solutions |
| PES membrane sterile syringe filters, 0.22 um, 30 mm | CELLTREAT | 229747 | for filtering solutions drawn into syringes |
| Petri dishes, 35 x 10 mm | Genessee Scientific | 32-103 | for micro dissection and to hold Tip dip solution in perforated-patch configuration |
| Petri Dishes, 60 x 15 mm | Midland Scientific | P7455 | for gross dissection |
| pH Meter | Mettler Toledo | Model S20 | |
| Pipettors (1000, 200, 10) microliter | USA Scientific | ||
| Poly-d-lysine coated glass bottomed culture dish | Mattek | P35GC-0-10-C | to plate neurons for culture |
| Quick change platform, heated base, for 35 mm culture dishes | Warner Instruments | 64-0375 | |
| Reference Cell | World Precision Instruments | RC1T | |
| Scalpel blade | Miltex | 4-315 | |
| Scalpel Handle | Fine Science Tools | 10003-12 | |
| Scientific Scale | Mettler Toledo | XS64 | |
| Serological Pipettes (10, 25) milliliter | Fisher Scientific | ||
| Silicone Grease Kit (for sealing coverglass and chamber) | Warner Instruments | 64-0378 | |
| Small Animal Guillotine | Kent Scientific | DCAP | |
| Small animal guillotine | Kent Scientific | DCAP | for decapitation if dissecting rats older than P15. |
| Stereo Dissection Microscope | Zeiss | Stemi 2000 | |
| Straight surgical scissors | Fine Science Tools | 14060-09 | |
| Syringe (3, 10, 30) milliliter | |||
| Trypsin | Sigma Aldrich | T1426 | one out of three enzyme to digest tissue |
| Tuberculin syringe | Covidien | 8881500105 | for delivering euthanasia solution by intraperitoneal injection |
| Vannas Spring Scissor, 2.5 mm Cutting Edge | Fine Science Tools | 15000-08 | |
| Volumetric flask, 1000 milliliter | |||
| Vortex | VWR | 945300 | |
| Water, sterile u ltrapure, R>18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system) | Millipore-Sigma | CDUFBI001 |
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Cite This Article
Iyer, M. R., Ventura, C., Bronson, D., Nowak, N., Regalado, K., Kalluri, R. Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings. J. Vis. Exp. (194), e64908, doi:10.3791/64908 (2023).