机械操纵控制神经元轴突的发展
Summary
应用在2-1000 microdyne范围内的神经元上的力量和直接测量是实现与高精密校准的玻璃针。这种方法可以用来控制和测量轴突发展的几个方面，包括轴突起始，轴突紧张，轴突伸长速度，和力矢量。
Abstract
细胞操作和神经轴突的延伸，可以校准玻璃微纤维能够测量和应用在10-1000μdyne范围1,2的力量来完成。力的测量是通过观察Hookean弯曲的玻璃针，这是直接和实证方法3校准获得。设备制造，校准，处理和使用的针头对细胞的要求和程序进行了全面介绍。力制度与以前使用的不同的细胞类型，这些技术已应用示范的方法的灵活性，并为今后的调查4-6的例子给出。技术优势是连续的“可视化”的操作所产生的力量和能力在各种细胞活动的直接干预。这些措施包括直接刺激和调节轴突生长和回缩 7，以及分队，对任何类型的培养细胞 8机械测量。
Protocol
1。使玻璃针。 一个可调的微针车夫是用来制造一个约4毫米长，已关闭了坚实的梁锥尖的针。相对于长期的灵活的提示，这短短的4毫米的长度将限制在实验过程中针尖的振动。在4毫米的光纤近端地区，针锥迅速从玻璃管的直径在1毫米至15微米，而最远端1毫米的纤维直径为2.5微米。我们使用的R – 6玻璃毛细管，外径为0.9毫米，编号0.6毫米8“长度和BB – CH车夫。如果研究者感兴趣的是在测?…
Discussion
技术应用和测量细胞的力量，有着悠久的历史9。我们的方法最初是由丹尼斯布雷，谁使用玻璃针，类似我们的“拖”的神经元在一个恒定的速率使用机动液压设备10的工作积极性。有许多细胞，其中包括应用力量的替代手段：步进电机11，磁珠12日，13微束和流体流动14。后者是类似于我们的做法，在蜂窝探头最终是对一个已知的刚度弯曲梁校准和蜂窝测量?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
在这种方法的发展，我们非常感谢博士罗伯特E巴克斯鲍姆的重要贡献。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| R-6 cap. Tube | Drummond Scientific Co., Broomall, PA, USA | 9-000-3111 | R-6 glass OD 0.9mm, ID 0.6 mm, 8″ | |
| BB-CH puller | Mecanex S.A., Geneva, Switzerland | BB-CH puller | Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000 | |
| 0.001″ Chromel wire | Omega Engineering, Stamford, CT, USA | SPCH-001-50 | unsheathed, themocouple wire, 50ft spool now called Chromega | |
| 0.003″ Constatan wire | Omega Engineering, Stamford, CT, USA | SPCI-003-50 | unsheathed, themocouple wire, 50 ft spool | |
| fine forceps | Fine Science Tools, USA | 91150-20 | Dumont Inox #5 | |
| universal microscope boom stand | Nikon | 76135 or 90430 | most brands or types of boom stand will work for this use | |
| mechanical micromanipulator | Narishige | M-152 | three-axis direct-drive coarse micromanipulator | |
| hydraulic micromanipulator | Narishige | MO-203 | now available as MMO-203, three movable axis type | |
| needle holder | Leica Microsystems | 11520145 | set of 3 | |
| single instrument holder | Leica Microsystems | 11520142 | ||
| double instrument holder | Leica Microsystems | 11520143 | ||
| mechanical micromanipulator | Leica Microsystems | 39430001 | post mount,1 prob holder, RH Model 430001 | |
| joystick mech. micromanipulator | Leica Microsystems | 11520137 | ||
| Leica DM IRB | Leica Microsystems | inverted microscope | ||
| Vibraplane isolation table | Kinetic System, Boston, MA, USA | 1200 series | ours is model 1201-02-12 | |
| Ringcubator | self manufactured see reference 19 | reference 19, requires updated controller listed below | ||
| programable temperature controller | Instrumart.com | Fuji Electric PXR3 | replaces the retired PXV3 temperature controller | |
| Nikon Diaphot TMD | Nikon Instruments, Inc. | inverted microscope, circa 1980 | ||
| Nikon SMZ-10 binocular dissecting | Nikon Instruments, Inc. | other dissecting microscopes will work |
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Tags
Mechanical ManipulationNeuronsControlAxonal DevelopmentGlass Micro-fibersForcesHookean BendingCalibrationEquipment RequirementsProceduresFabricatingTreatingCellsForce RegimesCell TypesFlexibilityMethodologyInvestigationVisualizationDirect InterventionCellular EventsStimulationRegulationAxonal GrowthRetractionDetachment
Citer Cet Article
Lamoureux, P., Heidemann, S., Miller, K. E. Mechanical Manipulation of Neurons to Control Axonal Development. J. Vis. Exp. (50), e2509, doi:10.3791/2509 (2011).