Name: design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits
Uploaded: 2015-04-15T13:00:00
Description: Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

这项工作是由欧洲项目BRIANBOW（FP7-年轻探险家的支持，笔者想感谢雅格布Tessadori博士对稿件有用的意见，和Silvia Chiappalone为她制作的视频所使用的图形帮助。

该程序是根据美国国立卫生研究院的护理标准和使用实验动物的完成，并批准了特拉维夫大学动物护理和使用委员会（许可证号 - L-14-019）。 1.准备仪器的PDMS <ol><li>准备晶片（ 材料的表 ，或从一个微细加工实验室的晶片），手术刀和镊子-灭菌是不需要的。 </li><li>根据以下条件使聚-D-赖氨酸（PDL）溶液：4毫克/毫升的0.1M硼酸盐缓冲液，pH 8，并储存在-20℃。 </l...

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的协议种植2D模块化神经网络的体外功能上相互连接组成的电路描述。该过程是基于图案的蜂窝的粘合剂层。图案形成用的PDMS模具再现期望网络体系结构的负特性来实现的。 PDMS模板定义，其中所述蜂窝粘合剂层沉积领域。一旦细胞被铺板，它们自发地装配到涂覆岛屿和自组织成活性间连接的电路。利用多边环境协定和钙成像记录功能模块化的网络录音提交。 所提出的...

实验和理论的证据支持，大脑运作通过的单元组件1-5协调活化，这可以被视为动态功能单元瞬时彼此，整形和下层不同大脑状态相互作用的可能性。官能模块化还依赖于与大脑电路6,7的结构的模块化组织关联。怎样的功能和大脑回路的结构互相塑造彼此仍是主要的开放性问题中的神经之一。以提供对这个问题更深入的了解，来识别最佳实验框架，其中能够解决，至少部分地，这些问题是重要的。由于控制操作的神经元网络的时空动力 ​​学在体内实验是具有挑战性的， 在体外的神经元网络模型的开发是显著感兴趣的，因为它们容易ACCessibility，监控，处理和建模8,9。在最近几年， 在由先进衬底图案化的方法支持体外技术已经允许诱导神经元网络开发一系列预定义的模块化结构体3，并与所施加的拓扑结构10学习网络的功能特性。特别是，方法进行了最近使用过的征收物理约束4,11组织网络。的确，以研究在神经元网络的结构和功能之间的联系，并提供相互作用的神经元组件的简化，但可能的表述， 在体外系统应提供相互连接的神经元亚群。广泛的研究二维均匀的神经细胞也没有对电路的自组织紧急连线任何空间的限制。因此，一个可能的方法来塑造人为地相互连接的电池组件被定位在不同的吐口水的神经元群体ially不同的领域。这些区域之间的距离不阻止除装配连接。这种方法，同时保证有相当的控制网络的复杂性，已经显示出，以提供同步模型6,7,12的更丰富的剧目。 为了方便的模块化神经元组件的可重复培养，协议组装的自组织网络到由轴突和树突连接的神经元集群的呈现和描述。聚合物结构对神经元培养物的物理约束已创建从polydimtheylsiloxane（PDMS）。 PDMS是一种弹性体广泛用于由于其生物相容性，透明性和透气性13生物医学应用。将PDMS制备并排除在微加工的SU8 2075 14,15的结构，通过旋涂液态PDMS上如杰克曼等人以前描述了“主”。16吨他实现了图形化的神经网络是由不同大小的相互连接的模块和他们两个盖玻片和微电极阵列（多边环境协定）17-20成功获得。在模块之间连接的密度可以改变它的网络同步的功能，从一个完全同步的网络中，典型的均一培养，以同步的瞬时状态模块之间。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PDMS, Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Vacuum Chamber</td>
			<td>Thermo</td>
			<td>5305-0609</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine PDL</td>
			<td>Sigma</td>
			<td>P7886</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell - Technologies Corporation</td>
			<td>WS-650-23</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well culture plate</td>
			<td>Sigma</td>
			<td>CLS3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8217;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA1060-Inv-BC</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Biological Industries Beit Haemek</td>
			<td>03-033-1c</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Minimum Essential Medium-Eagle</td>
			<td>Biological Industries Beit Haemek</td>
			<td>01-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Electrode Arrays 4Q</td>
			<td>Multi Channel Systems</td>
			<td>60-4QMEA1000iR-Ti-pr</td>
			<td>cleaning manual: http://www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>silicon wafer</td>
			<td>microchem</td>
			<td>SU8-2075</td>
			<td>Preparation protocol: www.microchem.com</td>
		</tr>
	</tbody>
</table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

大脑工作通过协调激活和神经元组件的动态通信。一个主要的开放问题是如何动态图案​​的巨大的剧目，这背后最多样化的大脑功能，可以浮现出的大脑回路的固定拓扑和模块化的组织。相比对神经元回路的体内研究，其呈现固有的实验的困难， 在体外制剂提供了更大的可能性，以操纵和探针实验神经系统的结构，动力学和化学性质。这项工作描述了在体外实验方法，它允许增长了空间层次分明，功能上相互联系的神经元组成的组件模块化网络。该协议允许控制神经网络的二维（2D）结构在不同级别的复杂性拓扑。 所期望的网络图案可定期盖玻片和基材嵌入式微电极阵列来实现两者。微机械结构被压印在硅晶片上，并用于创建生物相容性聚合物模具，其包含所述期望的网络架构的负特征。刻花模板与一个分子层用于促进细胞粘附的表面涂层过程期间放置在培养底物。除去模板后，神经元被镀和它们自发地重定向到涂覆区域。通过减小隔室间的距离，因此能够获得任何分离的或互相连接的神经元回路。促进细胞存活，细胞共培养，其位于培养皿的周围的支撑神经元网络。分别通过使用基板嵌入式微电极阵列和钙成像获得模块化网络的活动的电和光学记录被呈现。虽然每个模块显示SPONTaneous全球同步，对模块间同步的发生是由电路之间连接的密度调节。

甲SU8-2075模具的硅晶片具有约100μm的厚度的功能上是用来塑造的PDMS。的图案是由多个维度的平方，具有200至700微米（ 图1B）的侧的长度和距离变化。被选为广场的大小以适合的观点10X的字段（岛屿与一个边长&lt;800微米）和20X目标（对于岛屿边长&lt;400微米）。三个参数，电路之间即细胞铺板密度，距离，电路&#39;的大小是决定用于得到单层或聚集电路以及强加和形状的连接。然而，这?...

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

这手稿描述了一个协议， 在体外模块化网络包括空间上的限制，在功能上相互连接的神经回路的增长。的聚合物掩模被用来图案的蛋白质层，以促进通过培养基底细胞粘附。镀神经元生长在涂层区域自发建立连接并显示出电活性。

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Research

JoVE Journal

Neuroscience

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

测绘激光扫描Photostimulation抑制神经回路

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

科研

神经科学

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

重建神经元电路：快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...