神经活动的传播的展开海马准备具有穿透微电极阵列
Summary
我们已经开发了一种在体外展开海马其中保留神经元的CA1-CA3阵列。结合穿透微电极阵列，神经活性可以在纵向和横向取向来监测。这种方法提供优于海马切片制剂作为整个海马的传播可以同时记录。
Abstract
这个协议描述了用于制备新的体外平坦海马制备结合了微加工阵列映射神经活动在海马的方法。横向海马切片制备是最常见的组织制剂来研究海马电。纵向海马切片也被开发，以调查海马纵向连接。完整的小鼠海马也可以在体外保持，因为它的厚度允许足够的氧扩散。但是，这三个制剂不提供直接访问神经传播，因为一些组织是丢失或折叠。展开的完整海马提供横向和在一个平面配置为直接访问组织分析在体外海马信号传播的完全程度纵向连接。为了有效地监控从T的神经活动他细胞层，定制穿透微电极阵列（PMEA），制作并施加到展开的海马。为200微米的高度64的电极的PMEA可以记录的神经活动的小鼠海马深处。未折叠海马制备及PMEA的独特组合提供了一种新的体外工具来研究的速度和神经活动的传播方向在海马的二维CA1-CA3区具有高信噪比。
Introduction
理解的神经传导的神经信号或传播是用于判定在两个正常功能和病理条件神经通信在大脑1-3的机制是至关重要的。海马是最广泛研究的结构在大脑中，因为它起着基本作用在几个脑功能，如存储器和空间跟踪并参与几个病理变化，可显着地影响行为，以及1,6-之一。虽然，海马表现出复杂的组织，其结构的不同元件可以容易地确定和切片制备4-6访问。在海马的横向方向，神经活动已知通过三-突触通路组成该齿状回（DG），CA3，CA1 andsubiculum 4,5-传播。据认为，突触传递和轴突传导中发挥重要作用的通信对于在这横电路4,6。然而，神经信号的传播发生在横向和纵向方向上4,6。这意味着，海马，不能充分利用切片制剂限制观察到传播4的特定方向的影响。纵向切片的开发是为了调查沿着纵向轴线5的轴索途径。研究人员已经观察到的行为的特定r和θ振荡主要沿着横向和纵向轴线分别为6。这些行为已分别研究，但同时访问两个方向关键是要了解这些行为。甚至与完整海马制备的发展，它是很难监测在整个组织中传播，由于海马4的折叠结构。展开海马提供访问打包元在一个平面二维细胞层7,8的一种形式。
通过展开齿状回（DG）（ 图1），海马采用扁平形状具有矩形结构，其中横向和纵向的连接保持不变与设置在一个含两CA3和CA1二维片锥体细胞层，留下一块平坦的，可用于研究神经传播神经组织（ 图2）8。神经活动然后可以与个别玻璃吸管，微电极阵列，刺激电极，以及电压敏感染料（VSD）3,7,8监测。另外，从转基因小鼠的基因编码的电压指示器可用于跟踪传播图9。
展开的海马网络的扁平结构非常适合于光学记录方法，而且对于一个微电极阵列。 M市售阵列OST的制作扁平或低调的电极，可在组织切片和培养的神经元10-12记录的神经活动。然而，当信号从一个完整的组织中获得，因为神经元的胞体均位于更深的组织中的信号 – 噪声比（SNR）减小。具有高纵横比的微电极的电极阵列，需要改善的信噪比。
为此，一个穿透微电极阵列（PMEA）已经制定在我们的实验室，并通过插入64尖峰的直径为20微米和200微米的高度成折叠海马7,13-提供直接探测到组织的能力。此微电极阵列具有更高的SNR相比，电压敏感性染料成像和信噪比实验7,13期间保持稳定。展开的海马制备及PMEA的结合提供了一个投资新方式IGATE过的二维平面上的神经传播。使用这种技术的实验已经产生了关于神经信号传播在海马机制显著结果，由此神经活性可以独立的突触或突触电动7传播。
Protocol
Representative Results
Discussion
展开的海马制备，其中海马的纵轴和横轴被保存在具有穿透微电极阵列结合的发展，提供了一个有力的工具，调查解剖连接或神经传播在海马7。此展开过程也适用于在成年小鼠研究海马。与此制剂最近的研究表明，4-AP诱发的癫痫样活动可与一个对角波前越过展开的海马的整个区域（ 图6）7,8-传播。这些研究表明，完整的未折叠海马提供了超过任一横向或纵向切片显?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institutes of Health (National Institute of Neurological Disorders and Stroke) Grant 1R01NS060757-01 and by the E.L. Lindseth endowed chair to Dominique M. Durand. We thank Dr. Andrew M. Rollins’ laboratory for the help on the OCT imaging.
Materials
| desiccator jar | LABRECYCLERS Inc. | 5410 | Place regular paper towels at the bottome of the jar for animal anesthesia use. |
| A blade and Custome made surgical stage for unfolding hippocampus | N/A | N/A | A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it. |
| Custom made tissue recovery chamber | N/A | N/A | Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF. |
| Straight Operating Scissors | Fisher Scientific | S17336B Medco Instruments No.:81995 | This scissors is used to decapitate the mice. |
| Integra Miltex Goldman-Fox Scissors | Fisher Scientific | 12-460-517 MILTEX INC No.:5-SC-320 | This scissors is used to cut the skull of the mice. |
| Miltex Hysterectomy Forceps |
Claflin Medical equipment | CESS-722033-00001 | This Forceps is used to peel the cut skull to expose the brain |
| Micro Spatula | Cardinal Health | This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber. | |
| Frey Scientific Stainless Steel Semi-Micro Spatula | Cardinal Health | this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber. | |
| small paint brush | Lowe's | tem #: 105657 Model #: 90219 | The one with the smallest size in a normal paint brush package |
| Fire polished glass help tool | N/A | N/A | This tool was fire polished and made from the regular Pasteur glass pipettes. |
| Custom made glass needle | N/A | N/A | This tool was fire polished and made from the regular Pasteur glass pipettes. |
| Custom made glass tool with a metal wire loop | N/A | N/A | This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop. |
| Custom made glass solution dropper | N/A | N/A | This tool was made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end. |
| Custom made tissue anchor | N/A | N/A | Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator. |
| Custom fabricated microelectrode array | N/A | N/A | More detail about the array please refer to Kibler, et al, 2011. |
| Custom made filter and amplifiers circuits for the array | N/A | N/A | More detail about the array please refer to Kibler, et al, 2011. |
| Data acquisition processor 3400a | Microstar Laboratories | N/A | This is a complete data acquisition system with A/D converter. |
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