全细胞膜片钳红外神经刺激机制的调查
Summary
红外神经刺激已经提出作为一种替代电刺激神经类型，包括那些与听觉系统的范围内。本协议描述了膜片钳技术，在初级听觉神经元的文化学习机制的红外神经刺激。
Abstract
近年来已被证明在脉冲红外激光可用于诱发电反应在神经组织中，独立于目标组织的任何进一步的修改。红外神经刺激已报道在体内的各种外设和感觉神经组织中，特别令人感兴趣的听觉神经的神经元的刺激中所示。但是，而INS已被证明在这些设置，由红外光引起的神经兴奋的机制（或机制）目前没有得到很好的理解。这里介绍的协议描述了全细胞膜片钳的方法设计，以方便调查红外培养初级听觉神经元的神经刺激。彻底红外激光照射在受控条件下在体外 ，这些细胞的特征的响应，它可能是未能获得的基本物理之后更好地理解l和相关的生化过程红外神经刺激。
Introduction
神经生理学和医学仿生学领域倚重允许可控刺激的电反应神经组织的技术的。虽然电刺激神经 兴奋的金标准，它受到来自一些缺点，如刺激工件的存在下录音时的神经反应，和缺乏刺激特异性的电流的扩散到周围组织1。
过去二十年的发展已经看到光学介导的刺激技巧2。这些技术中需要几个靶组织的变形，可通过另外一个特定的分子（ 如笼分子）3或某种形式的遗传操作（ 例如，光遗传学）4，这两者都不容易申请以外的研究设置。因此特别令人感兴趣的是红外神经刺激（INS），whereb的Ÿ神经组织兴奋红外激光脉冲。 INS有可能克服许多电刺激的缺点，通过使具有高度特异性，非接触刺激神经 组织2。然而，虽然INS已成功地展示了在体内的各种设置，激励的准确机理仍然不明朗。
最近的一些出版物揭露背后的机制INS 5-7所示的进展。由于吸收的激光水快速加热，似乎发挥了关键作用。然而，除了尚未达成共识。 Shapiro 等人 7提出了一种高度的通用性的机制，从而快速加热导致的扰动，在细胞膜上相邻的带电粒子的分布，导致细胞膜和随后的去极化的电容的变化。此外，阿尔伯特等 5断言LASER明显加热温度敏感离子通道（瞬时受体电位香草频道）激活一个特定的类，允许离子通过细胞膜。在这个阶段，目前还不清楚如何将这些机制相结合，或什至是否有进一步的因素，这些因素尚未确定。
调查虽然数量不多的出版物（5,7-9引用）INS 体外发表在这一领域的工作，绝大多数已进行了体内 （ 如引用1,6,10-18）。红外刺激听觉神经元已经特别感兴趣的区域，由于人工耳蜗植入10,14-18中的潜在应用。 在体内实验中是很重要的验证技术在各种设置中， 在体外研究中所提供的控制水平增加，预计将导致一个更详细的了解机甲的有效性负责INS anism。这份报告介绍了编制的螺旋神经节神经元膜片钳调查，因为这些可以用来研究的基本机制，同时也连接到现有的数据从听觉系统的庞大的身躯。
膜片钳技术电现象的调查是一个很好的工具，它提供了一种在单细胞记录电活动和研究的贡献，个人的基本电流19。 在体外制备，如螺旋神经节神经元的初级神经元，当这项技术被应用到一个稳定的，它提供了学习的机会，在深度神经活动的机制，控制和操纵。
在这项工作中轮廓的方法调查激光刺激螺旋神经节神经元对电性能的影响，通过膜片钳指定的协议录音。该方法是基于在光纤耦合激光，而不是一个自由空间激光，允许更安全的操作，以及更容易，更可重复的对齐，而不需要修改标准的显微镜配置。这些协议的基础上，它应该是可能的，为了更清楚地确定的机制或机制背后INS进行了广泛的实验。
Protocol
1。螺旋神经节神经元的文化消毒小轮（ 如 10毫米直径）盖玻片，在高压釜中弯钳。无菌盖玻片转移到无菌的4 – 环35毫米陪替氏培养皿或4 – 孔板的各井，用无菌镊子。应用150μl的聚-L-鸟氨酸（500微克/毫升），小鼠层粘连蛋白（0.01毫克/毫升）在盖玻片和地点的培养箱中（37℃）的顶表面48小时。确保盖玻片不浮离井底。 准备50毫升无菌的Neurobasal培养基（NBM）的每个神经培养?…
Representative Results
螺旋神经节神经元激光照射可重复波形电压钳和电流钳记录配置中， 图3a示出典型的电流的变化，响应穿过细胞膜的一个2.5毫秒，0.8毫焦耳的激光脉冲（平均响应于6激光脉冲，在1秒的间隔）与膜电位在-70mV，-60 mV和-50 mV的重复。净内向电流一贯诱发在激光脉冲响应，停止光照后返回到初始值。可以看出，激光诱导的电流的形状，以改变膜电位改变，这表明它可能是重要的，在钳制电?…
Discussion
使用本文中列出的协议，它可以提取和培养螺旋神经节神经元，并探讨激光诱发的电活动进行全细胞膜片钳实验。 在体外使用时，膜片钳技术提供实验参数的控制，是无法实现在体内的水平。激光刺激参数，如波长，脉冲能量，脉冲长度，脉冲形状和脉冲重复序列，可以研究在一个可重复的设置。此外，维持神经元所处的环境（ 例如，溶液的温度，化学因素）可以系统地改?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由澳大利亚研究理事会根据联动项目赠款LP120100264支持。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Cell culture materials and equipment | |||
| Glass coverslips | Lomb Scientific | CSC 10 1 GP | |
| 4-ring cell culture dish | VWR International | 82050-542 | |
| Poly-L-ornithine solution | Sigma-Aldrich | P4957 | |
| Laminin | Invitrogen | 23017-015 | |
| Curved forceps | WPI | 14101 | Dumont #5 tweezers (45° angle tip) |
| CO2 Incubator | ThermoScientific | Heracell 150i | |
| Table 1. Cell culture materials and equipment. | |||
| Neurobasal media | |||
| Neurobasal A | Gibco | 10888-022 | |
| N-2 supplement | Invitrogen | 17502-048 | |
| B27 serum-free supplement | Invitrogen | 17504-044 | |
| Penicillin-Streptomycin | Invitrogen | 15140-148 | |
| L-Glutamine | Invitrogen | 25030-149 | |
| Intracellular solution | |||
| Potassium chloride | Sigma-Aldrich | P4504 | |
| HEPES | Sigma-Aldrich | H4034 | |
| Potassium D-gluconate | Sigma-Aldrich | G4500 | |
| EGTA | Sigma-Aldrich | E3889 | |
| Na2ATP | Sigma-Aldrich | A2383 | |
| MgATP | Sigma-Aldrich | A9187 | |
| NaGTP | Sigma-Aldrich | G8877 | |
| Potassium hydroxide | LabServ | BSPPL738.500 | |
| Sucrose | Sigma-Aldrich | S8501 | |
| Extracellular solution | |||
| Sodium chloride | Sigma-Aldrich | 310166 | |
| Potassium chloride | Sigma-Aldrich | P4504 | |
| HEPES | Sigma-Aldrich | H4034 | |
| Calcium chloride | Sigma-Aldrich | 383147 | |
| Magnesium chloride | Sigma-Aldrich | M8266 | |
| D-Glucose | Sigma-Aldrich | G8270 | |
| Sodium hydroxide | LabServ | BSPSL740.500 | |
| Sucrose | Sigma-Aldrich | S8501 | |
| Table 2. Solutions for cell culture and patch clamp. a) Neurobasal media. b) Intracellular solution. c) Extracellular solution. | |||
| Upright microscope | Zeiss | AxioExaminerD1 | Equipped with Dodt contrast |
| Water-immersion objective | Zeiss | W Plan-APOCHROMAT 40x/0.75 | |
| Platform and X-Y stage | ThorLabs | Burleigh Gibraltar | |
| Recording chamber | Warner Instruments | RC-26G | |
| Vibration isolation table | TMC | Micro-g 63-532 | |
| CCD Camera | Diagnostic Instruments | RT1200 | |
| Camera software | Diagnostic Instruments | SPOT Basic | |
| In-line solution heater | Warner | SH-27B | |
| Temperature controller | Warner | TC-324B | |
| Patch clamp amplifier | Molecular Devices | Multiclamp 700B | |
| Patch clamp data acquisition system | Molecular Devices | Digidata 1440A | |
| Micromanipulator | Sutter Instruments | MPC-325 | |
| Micropipette glass | Sutter Instruments | GBF100-58-15 | Borosilicate glass with filament |
| Micropipette Puller | Sutter Instruments | P2000 | |
| Recording Software | AxoGraph | Lab pack and electrophysiology tools | |
| Aspirator bottle | Sigma-Aldrich | CLS12201L | 1 L Pyrex aspirator bottle, with outlet for tubing |
| PE Tubing | Harvard | PolyE #340 | |
| Masterflex peristaltic pump | Cole-Parmer | HV-07554-85 | |
| Table 3.Patch clamp equipment. | |||
| 1,870 nm laser diode | Optotech | ||
| 200/220 μm diameter multimode optical fiber patch cord (FC/PC) | AFW Technologies | MM1-FC2-200/220-5-C-0.22 | Light delivery optical fiber, silica core and cladding, 0.22 NA |
| Optical fiber through connector (FC/PC) | Thorlabs | ADAFC2 | |
| Optical fiber cleaver | EREM | FO1 | |
| Optical fiber stripping tool (0.25 – 0.6 mm) | Siemens | For removing optical fiber jacket | |
| Optical fiber stripping tool (0.6 – 1.0 mm) | Siemens | For removing outer coating of patch cord | |
| Signal generator | Any signal generator that can output the necessary pulse shapes and is capable of being externally triggered | ||
| Optical fiber positioner | Custom made positioner. Could substitute with standard micropositioner used for patch clamp experiments | ||
| Optical fiber chuck | Newport | FPH-DJ | |
| Laser power meter and detector head | Coherent | FieldMate (power meter) with LM-3 (detector head) | |
| Table 4. Laser equipment. |
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Citar este artigo
Brown, W. G. A., Needham, K., Nayagam, B. A., Stoddart, P. R. Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation. J. Vis. Exp. (77), e50444, doi:10.3791/50444 (2013).