爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议
Summary
本文讨论了三种用于全细胞膜片钳记录的脑准备, 研究了爪蟾蝌蚪的 retinotectal 电路。每个准备, 以它自己的具体优势, 贡献的实验驯良的爪蟾蝌蚪作为模型, 研究神经回路功能。
Abstract
爪蟾蝌蚪 retinotectal 电路, 由眼睛中的视网膜神经节细胞 (RGCs) 组成, 直接在视神经顶盖神经元上形成突触, 是研究神经回路自组装的流行模型。能够从 tectal 神经元进行完整的细胞修补钳录音, 并记录研资局诱发的反应, 无论是在体内还是使用全脑准备, 都产生了大量高分辨率的数据, 关于正常的机制、异常、电路的形成和功能。在这里, 我们描述如何执行在体内准备, 原来的整个大脑准备, 和最近开发的水平脑切片准备, 以获得整个细胞补丁钳录音从 tectal 神经元。每个制剂都具有独特的实验优势。通过体内准备, 可以记录 tectal 神经元对投射到眼睛上的视觉刺激的直接反应。整个大脑的准备, 使研资局的轴突被激活的高度控制的方式, 和水平脑切片准备允许记录从所有层面的顶盖。
Introduction
retinotectal 电路是两栖视觉系统的主要组成部分。它由眼睛中的 RGCs 组成, 它将它们的轴突投射到视神经顶盖, 与突触后 tectal 神经元形成突触连接。爪蟾蝌蚪 retinotectal 电路是研究神经回路形成和功能的一种流行的发展模式。这个蝌蚪的 retinotectal 电路有许多属性, 使它成为一个强大的实验模型1,2,3。这篇文章的主要特点之一, 是能够从 tectal 神经元、体内或使用整个大脑准备进行完整的细胞贴片钳录音。有一个电生理学钻机配备了一个放大器, 支持电压和电流钳记录模式, 整个细胞补丁钳录音允许神经元的电生理学的特点是高分辨率。因此, 整个细胞补丁钳录音从 tectal 神经元跨越关键阶段的 retinotectal 电路形成提供了详细和全面的理解的发展和可塑性的内在4,5,6,7和突触8,9,10,11属性。结合全细胞贴片钳 tectal 神经元记录, 表达基因或 morpholinos 在这些神经元中感兴趣的能力12, 以及通过已建立的视觉避免测试 (13 ) 来评估视觉引导行为的方法, 可促进识别分子、电路功能和行为之间的链接。
重要的是要注意的是, 从整个细胞膜片钳录音获得的高分辨率数据的类型是不可能使用新的成像方法, 如基因钙指示器 GCaMP6, 因为虽然使用钙指标允许成像在大量神经元的钙动力学同时, 没有直接或明显的方法可以通过测量胞体中的三角洲荧光来获得特定的电学参数, 也没有办法对神经元进行电压钳位测量。电流-电压关系。显然, 这两种截然不同的方法, 电生理记录和钙成像, 具有不重叠的优势和产生不同类型的数据。因此, 最好的方法取决于正在处理的具体实验问题。
在这里, 我们描述了我们的方法, 从蝌蚪光学顶盖神经元, 使用体内准备, 全脑准备, 和一个新的修改全脑准备, 在我们的实验室中开发的全细胞补丁钳录音 14 .在 “代表性结果” 部分中, 我们演示了每个准备的实验优势和可以获得的不同类型的数据。讨论部分包括了不同准备工作的极限和强度, 以及排除故障的提示。
Protocol
这里描述的所有方法都已被怀俄明州大学的机构动物护理和使用委员会 (IACUC) 批准。所有的程序, 包括电生理记录, 都是在室温下进行的, 大约23摄氏度。这里描述的所有方法都是针对在发育阶段42和 49 (根据 Neiuwkoop 和费伯15) 的蝌蚪记录 tectal 神经元进行优化的。 1.体内准备 麻醉蝌蚪。 将蝌蚪放在含有斯坦伯格溶液的小培养皿中, 0.01% MS-222…
Representative Results
为了记录光诱发的反应, 整个领域的闪光投射到视网膜上, 而由此产生的响应是从单个的 tectal 神经元 (图 4a) 记录下来的。这个特殊的协议旨在测量神经元对光的响应 (“on” 响应), 然后在十五年代关闭以测量 “关闭响应”。Tectal 神经元通常表现出强健的和关闭的响应 (这里显示的电压钳模式, 与神经元钳位 60mV, 以测量突触电流 (<strong class="xfig…
Discussion
这项工作中描述的所有方法都是为记录发育阶段42和 49 (根据 Neiuwkoop 和费伯15) 的蝌蚪的 tectal 神经元而优化的。在阶段 42, 蝌蚪是足够大和充分开发的, 以便昆虫别针可以被安置在脑子的任一侧为在体内录音和进行整个脑解剖。在早期阶段, 当蝌蚪本质上是二维 (即, 平), 这里描述的方法是不最佳的。
由于 tectal 神经元可以在整个细胞配置中被访?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
由 NIH 赠款 SBC COBRE 1P20GM121310-01 支持。
Materials
| Stemi Stereo 508 | Zeiss | 495009-0006-000 | Dissecting microscope |
| MS-222 "Tricane" | Finquel | ARF5G | Amphibian general anesthetic |
| Sodium Chloride (NaCl) | Fisher Scientific | S271-3 | Used to prepare Stienberg's solution and external solution |
| Potassium Chloride (KCl) | Fisher Scientific | P217-500 | Used to prepare Stienberg's solution and external solution |
| HEPES | Sigma-Aldrich | H3375-1KG | Used to prepare Stienberg's solution and external solution |
| Calcium nitrate tetrahyrate (Ca(NO3)•4H2O) | Sigma-Aldrich | 237124-500G | Used to prepare Stienberg's solution |
| Magnesium Sulfate (MgSO4) | Mallinckrodt Chemicals | 6066-04 | Used to prepare Steinberg's solution |
| Calcium Chloride (CaCl2) | Sigma-Aldrich | C5080-500G | Used to prepare external recording solution |
| Magnesium Chloride (MgCl2) | J.T. Baker | 2444-01 | Used to prepare external recording solution |
| D-glucose Anhydrous | Mallinckrodt Chemicals | 6066-04 | Used to prepare external recording solution |
| Tubocurarine hydrochloride pentahydrate | Sigma | T2379 | Nicotinic acetylcholine receptor antagonist |
| Insect Pins | Fine Science Tools | 26002-10 | 0.1mm diameter stainless steel pins |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | 761028 | Preweighed monomer and curing agent kit |
| Sterile Polystyrene Petri Dish – 60x15mm | Fisher Scientific | AS4052 | Small petri dishes |
| PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm) | BD | 305122 | Syringe needles |
| 1mL Slip Tip Tuberculin Syringe | BD | 309659 | Disposable, sterile syringes |
| Borosilicate pipette glass | Sutter Instrument | BF150-86-10HP | Pulled to desired specifications using pipette pulling machine |
| Flaming/Brown Micropipette Puller | Sutter Instruments | P-97 | Fabricates micropipettes for electrophysiology recording |
| Kimwipes Kimtech wipes | Kimberly-Clark | 34120 | Delicate task lint-free wipers |
| Axon Instruments MultiClamp 700B Headstage CV-7B | Molecular Devices | 1-CV-7B | Current clamp and voltage clamp headstage |
| MP-285 Motorized Manipulator with Tabletop Controller | Sutter Instrument | MP-285/T | Control for headstage on electrophysiology rig |
| Fiber-Coupled LED (Green) | Thorlabs | M530F2 | Fiber optic cable paired with green LED |
| Cluster Bipolar Electrode (25µm diameter) | FHC | 30207 | Bipolar stimulating electrode |
| ISO-Flex Stimulator | A.M.P.I. (Israel) | Contact manufacturer | Flexible stimulus isolator |
| Axon Instruments 700B Multipatch Amplifier | Molecular Devices | 2500-0157 | Amplifier for voltage- and current-clamp recording |
| Digidata 1322A digitizer | Molecular Devices | 2500-135 | Data acquisition system for electrophysiology recording |
| Axio Examiner.A1 | Zeiss | 491404-0001-000 | Microscope for electrophysiology |
| Micro-g Lab Table | TMC | 63-533 | Air table for electrophysiology microscope |
| Inspiron 620 Personal Desktop Computer with Windows 7 64-bit | Dell | D06D001 | Computer running electrophysiology software |
| c2400 CCD camera | Hamamatsu | 70826-5 | Charge-coupled device camera for electrophysiology imaging |
| 7 O'Clock Super Platinum Stainless Razorblades | Gillette | CMM01049 | Platinum-coated stainless razor blades |
| Transfer Pipets | Fisher Scientific | 13-711-7M | Disposable Polyethylene transfer pipets |
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Citazione di questo articolo
Liu, Z., Donnelly, K. B., Pratt, K. G. Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons. J. Vis. Exp. (133), e57465, doi:10.3791/57465 (2018).