使用电动和一维和二维文化磁场神经元的外部激励
Summary
神经元培养是通过其对单个神经元或神经元群体的影响来研究新兴脑刺激技术的良好模型。这里提出的是通过直接由浴电极产生的电场或由时变磁场诱导的电场刺激图案化神经元培养物的不同方法。
Abstract
当它的膜电位超过某个阈值的神经元就会触发动作电位。在大脑中的典型的活性，这发生如化学投入到它的突触的结果。然而，神经细胞也可以通过强加的电场激发。尤其是，最近的临床应用程序激活由外部产生电场的神经元。因此，感兴趣的研究神经元如何响应外场是什么原因导致的动作电位。幸运的是，外部电场的精确和受控应用是可能的被切下，解离和在培养物中生长的神经元的胚胎细胞。这使得在一个高度可重复的系统，这些问题的调查。
在本文中的一些用于外部电场对神经元培养物控制的应用程序的技术进行了综述。这些网络可以是一维的，在LINEA 即图案化ř形式或允许在基板的整个平面成长，因此二维的。此外，激发可通过电场直接应用通过浸入流体（浴电极）或通过使用磁脉冲的远程创建诱导电场的电极产生。
Introduction
神经元和外部电场之间的相互作用具有根本性的影响以及实际的。虽然人们自从沃尔的倍外部施加的电场可以激发组织已知的，负责神经细胞中生产的所得动作电位的机制最近才开始被揭开1，2，3，4。这包括关于查找导致膜电位的去极化，膜性质和离子通道的作用，甚至该区域响应该电场2,5神经元的机制问题的答案。治疗神经刺激6，7，8，9， <sup10类方法特别依赖于这些信息，这对于针对受影响的地区和了解治疗结果来说可能至关重要。这种理解也可以帮助制定治疗方案和刺激大脑不同区域的新方法。
测量体内大脑内的相互作用为这一理解提供了重要的组成部分，但受到颅骨内测量不精确和低可控性的阻碍。相比之下，文化中的测量可以很容易地以高精度，优异的信噪比和高重现性和控制的高精度执行。使用文化可以阐明集体网络行为的各种神经元属性11,12,13,14 ， </sup> 15，16 。类似地，期望这种良好控制的系统在阐明其他刺激方法的作用机制方面是非常有效的，例如在光激活神经元17,18,19中的光学刺激期间如何通道开放负责产生动作电位。
这里的重点是描述可以通过外部电场有效地激发神经元的工具的开发和理解。在本文中，我们描述了二维和一维图案海马培养物的制备，使用浴电极直接施加的电场的不同构型和取向的刺激，最后通过一个刺激二维和图案化的一维培养物时变磁场，其引起电场5，20，21 。
Protocol
伦理声明：涉及动物的处理程序是按照魏茨曼科学研究所的机构动物护理和使用委员会（IACUC），以及适当的以色列法律的指引完成。魏兹曼研究所由协会为实验动物国际评估和认可委员会（AAALAC）的认可。魏茨曼机构动物护理和使用委员会批准了这项研究，与海马神经元进行。 1.二维（2D）和一维（1D）海马培养制备盖玻片2D培养的制备。 制备平板培养基（PM）…
Representative Results
提出的协议允许神经元培养物的容易构图。当它与我们为刺激开发了几种方法相结合，它能够使一些内在的神经元属性，如时值和Rheobase 5的测量，比较健康和疾病的神经元27的特性，找到最佳的方法来刺激文化之间的函数关系它们的结构和更多的新方法。一些例子，在未来的数字呈现。 <p class="jove_content" fo:keep-together.within-page="1"…
Discussion
1D图案是可用于多种应用的重要工具。例如，我们已经使用一维图案用于从神经元培养29创建逻辑门以及最近测量大鼠海马神经元5的时值和Rheobase，和唐氏综合征的海马神经元电活动的信号传播速度的下降的减速比野生型（WT）海马神经元27。一维图案的建议的方案是稳健的，这是容易形成任何所需的图案。我们建议观察1D培养前测量，以了解?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢奥弗·费纳曼，弗雷德·沃尔夫，梅纳姆·塞加尔，安德烈亚斯·尼夫和艾塔恩·雷威尼非常有益的讨论。作者感谢伊兰·布雷斯金和佐迪·索利安诺开发技术的早期版本。作者感谢茨维·拉斯蒂和吉恩·皮尔·埃克曼与理论概念的帮助。这项研究是由密涅瓦基金会，科技，以色列，部和以色列科学基金会授予1320年至1309年和碧国家科学基金会资助2008331支持。
Materials
| APV | Sigma-Aldrich | A8054 | Disconnect the network. Mentioned in Section 2.4.2 |
| B27 supp | Gibco | 17504-044 | Plating medium. Mentioned in Section 1.1.1 |
| bicuculline | Sigma-Aldrich | 14343 | Disconnect the network . Mentioned in Section 2.4.2 |
| Borax (sodium tetraborate decahydrate) | Sigma-Aldrich | S9640 | Borate buffer. Mentioned in Section 1.1.2 |
| Boric acid | Frutarom LTD | 5550710 | Borate buffer. Mentioned in Section 1.1.2 |
| CaCl2 , 1M | Fluka | 21098 | Extracellular recording solution . Mentioned in Section 1.5.2 |
| CNQX | Sigma-Aldrich | C239 | Disconnect the network . Mentioned in Section 2.4.2 |
| COMSOL | COMSOL Inc | Multiphysics 3.5 | Numerical simulation. Mentioned in Section 3.5.2 |
| D-(+)-Glucose, 1M | Sigma-Aldrich | 65146 | Plating medium, Extracellular recording solution . Mentioned in Section 1.1.1 1.5.2 |
| D-PBS | Sigma-Aldrich | D8537 | Cell Cultures. Mentioned in Section 1.2.4 1.2.6 |
| FCS(FBS) | Gibco | 12657-029 | Plating medium. Mentioned in Section 1.1.1 |
| Fibronectin | Sigma-Aldrich | F1141 | Bio Coating. Mentioned in Section 1.2.6 |
| Fluo4, AM | Life technologies | F14201 | Imaging of spontaneous or evoked activity . Mentioned in Section 1.5.1 1.5.3 1.5.5 |
| FUDR | Sigma-Aldrich | F0503 | Changing medium. Mentioned in Section 1.4.1 |
| Gentamycin | Sigma-Aldrich | G1272 | Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1 |
| GlutaMAX 100X | Gibco | 35050-038 | Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1 |
| Hepes, 1M | Sigma-Aldrich | H0887 | Extracellular recording solution . Mentioned in Section 1.5.2 |
| HI HS | BI | 04-124-1A | Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1 1.4.1 1.4.2 |
| KCl, 3M | Merck | 1049361000 | Extracellular recording solution. Mentioned in Section 1.5.2 |
| Laminin | Sigma-Aldrich | L2020 | Bio Coating. Mentioned in Section 1.2.6 |
| MEM x 1 | Gibco | 21090-022 | Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1 1.4.2 |
| MgCl2 , 1M | Sigma-Aldrich | M1028 | Extracellular recording solution. Mentioned in Section 1.5.2 |
| NaCl, 4M | Bio-Lab | 19030591 | Extracellular recording solution . Mentioned in Section 1.5.2 |
| Octadecanethiol | Sigma-Aldrich | 01858 | Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3 |
| Pluracare F108 NF Prill | BASF Corparation | 50475278 | Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4 1.2.6 |
| Poly-L-lysine 0.01% solution | Sigma-Aldrich | P47075 | Promote cell division. Mentioned in Section 1.1.4 |
| Sucrose, 1M | Sigma-Aldrich | S1888 | Extracellular recording solution . Mentioned in Section 1.5.2 |
| Thiol | Sigma-Aldrich | 1858 | Bio-Rejection Coating. Mentioned in Section 1.2.3 |
| URIDINE | Sigma-Aldrich | U3750 | Changing medium. Mentioned in Section 1.4.1 |
| Sputtering machine | AJA International, Inc | ATC Orion-5Series | coating glass with thin layers of metal. Mentioned in Section 1.2.2 |
| Pen plotter | Hewlett Packard | HP 7475A | Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5 |
| Electrodes wires | A-M Systems, Carlsborg WA | 767000 | Electric stimulation of neuronal cultures. Mentioned in Section 2.1 2.2 2.3 2.4.5 |
| Signal generator | BKPrecision | 4079 | Shaping of the electric signal. Mentioned in Section 2.3 |
| Amplifier | Homemade | Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3 | |
| Power supply | Matrix | MPS-3005 LK-3 | Power supply to the sputtering machine. Mentioned in Section 1.2.2.3 |
| Transcranial magnetic stimulation | Magstim, Spring Gardens, UK | Rapid 2 | Magnetic stimulation of neuronal culture. Mentioned in Section 3.1 3.3 3.4 |
| Epoxy | Cognis | Versamid 140 | Casting of homemade coils. Mentioned in Section 3.4 |
| Epoxy | Shell | EPON 815 | Casting of homemade coils. Mentioned in Section 3.4 |
| Platinum wires 0.005'' thick; A-M Systems, | Carlsborg WA | 767000 | Electric stimulation of neuronal cultures. Mentioned in Section 2.1 |
| Circular magnetic coil | Homemade | Magnetic stimulation of neuronal culture. Mentioned in Section 3.3 | |
| WaveXpress SW | B&K Precision | Waveform editing software. Mentioned in Section 2.1.32 | |
| Xion Ultra 897 | Andor | Sensitive EMCCD camera. Mentioned in Section 2.4.4 |
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Tags
Keywords:
Neuronal CulturesElectric StimulationMagnetic StimulationOne-dimensional CulturesTwo-dimensional CulturesNeural ExcitationChronaxieRheobasePatterned Neuronal NetworksElectric FieldsMagnetic FieldsBrain DisordersCell CultureSputteringBiorejecting LayerChromeGoldPen PlotterEtching NeedleRubber Sheet
Citar este artigo
Stern, S., Rotem, A., Burnishev, Y., Weinreb, E., Moses, E. External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures. J. Vis. Exp. (123), e54357, doi:10.3791/54357 (2017).