Name: neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array
Uploaded: 2015-03-27T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array at JoVE.com

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&#8217; laboratory for the help on the OCT imaging.

注:动物实验方案进行了审查，并在大学批准的机构动物护理和使用委员会。 CD1小鼠任一性别的P10的年龄到P20被用于本研究。 1.解决方案手术和实验记录<ol><li>制备含有（mM计）正常的人工脑脊液（ACSF）缓冲液:氯化钠124，氯化钾3.75，KH 2 PO 4 1.25，MgSO 4干燥2， 碳酸氢钠 26，葡萄糖10，和氯化钙 2。在实验开始时使用该正常脑脊液解剖后组织的恢复，以及用于洗涤系统。 </li><li>备是海马解剖过程中使用，并含有蔗糖学联（mM计）:蔗糖220，氯化钾2.95，将NaH 2 PO 4 1.3，MgSO 4干燥2， 碳酸氢钠 26，葡萄糖10，和氯化钙 2。为了诱导癫痫样活动的完整海马组织制剂中，添加4-氨基吡啶（4-AP），以正常的脑脊液在为100μM的浓度。 </li></ol> 2.手术过程的完整海马准备<ol><li>滴异氟烷（1毫升）到腔室，在一个干燥器玻璃瓶（1号中的具体材料和设备）的底部，并使用常规的纸巾覆盖底部阶段的表面上，从而对动物没有得到与接触的液体。然后，将CD1鼠标放进瓶子里，并盖上盖子。保持在封闭罐的动物为约1分钟至2分钟。当呼吸频率为约1每秒，从罐取出鼠标。 </li><li>放置在鼠标上的手术阶段和斩首与合适的剪刀。立即斩首后，将头部放入冰冷（3-4℃），氧化蔗糖脑脊液约30秒。 </li><li>用细剪刀（第5号中的具体材料和设备），以去除皮肤上的头骨顶部和切割沿头部的中间线颅骨以及在接近速度两端RAL叶。使用钳子（6号中具体的材料和设备）剥离朝向头部的每一侧的切口颅骨以暴露脑。 </li><li>插入一个微型实验室压舌板（在特定的材料和设备第8号）到大脑和颅骨的顶叶之间的差距从头骨底部小心剥离脑，然后将其放到准备好的冰冷（3 -4°C）手术舞台铺上湿滤纸（第2号的具体材料和设备）。除去用冰 - 冷却的刀片小脑和通过切割脑的中线分开的两个半球。然后，将两个分离的半球入填充有冰冷蔗糖脑脊液通入95％O 2/5％CO 2的烧杯中。 </li><li>取一半的半球和地点上的冰冷滤纸阶段。将普通纸巾或滤纸周围脑吸多余的解决方案。使用两个火抛光的玻璃吸管工具（第10号中的材料和设备）与皮质从大脑的中心部分的其余部分分离。 </li><li>之后，海马从皮层内部暴露，换上了冰冷的阶段，将两三滴对组织冰冷的蔗糖学联然后取出海马有多余的解决方案。切断与皮层连接在海马的两端。然后，解剖海马出大脑与火抛光玻璃工具并取出大脑的剩余部分。 </li><li>分开整个海马与alveus面朝上，海马沟朝下。迅速下降两个或三个滴冰冷蔗糖脑脊液在组织再次并取出组织周围使用了一块纸巾的额外溶液。使用火抛光的玻璃的工具改过整个海马，露出沟（ 图1B，C）。 </li><li>在一个正常的光学显微镜，插入一个定制的玻璃针（11号在特殊材料 -ALS和设备）插入沟的一端，并切断光纤连接，从齿状回（DG）到下托或海马CA1领域，沿沟的方向（ 图1C）。适用的冰冷刀片如果需要修剪海马的间隔和时间结束。插入定制金属线环（第12号中的具体材料和设备）插入切沟，并从组织，同时保持了海马的下脚/ CA1端通过一火抛光的玻璃刀具拉过对DG远（ 图1D） 。 </li><li>按照上述的展开过程中，添加另外的两个或三滴的冰冷蔗糖脑脊液的到组织并移除组织周围的多余溶液。然后，修剪展开海马用冰冷刀片的边缘（ 图1E）和在制备用刮刀放入填充有正常脑脊液和通入95％O 2/5％的CO 2在室温下将回收室（约25℃）。离开展开海马组织，以将其放置到录音室之前收回约1小时。 </li><li>采取其他脑半球并用它要经过的步骤，从2.5到2.9。通常，需要约1至2分钟，以完成整个过程展开单个海马和连续地滴冰冷蔗糖脑脊液，以保持氧化和水合的组织。 </li></ol> 3.实验系统设置<ol><li>胶上的引脚栅格阵列（PGA）封装的定制制造的PMEA，并使用微引线接合到从一个单一的微小电极连接每个垫的包（ 图3B）的销的垫。然后，将包插入一个特制的电路板13的插座中。 </li><li>每个单独的微电极与带通截止频率为1赫兹的100上的定制的电路板13的增益连接到它的过滤器，以4千赫和放大器。由A / D转换数字化从电路板输出的模拟耳听系统，并获取和存储数据的计算机上。 </li><li>胶与入口和出口管，用于将溶液的流动（ 图4A）的阵列周围一个特制的塑料记录室中。使用至少两个瓶子，以保持不同的解决方案，在系统中，并加入并通过三阀控制瓶子的输出管道。连接三阀的一个管件系统的输出与静脉滴注室被引导溶液到记录室中。 </li><li>三阀和录音室的入口之间，加电加热器来加热该溶液到受控温度（35℃）之前的溶液被引导到记录室中。附着在记录室的出口到一个真空管收集溶液成真空管连接烧瓶中。不回收任何实验在这项研究中的任何解决方案。 </li></ol> 4.放置展开海马到PMEA录制的神经活动</p&gt; <ol><li>要准备的实验装置，填补一瓶正常脑脊液和另一瓶与4-AP学联。在这两个瓶，泡沫的95％ 的 O 2/5％CO 2，从各实验的开始。使用三 - 阀连接器，以控制该溶液将在实验中进行选择。连接一个真空管，在该腔室的出口到泵将溶液到贮尘容器。递送它到记录室之前加热该管道，并保持溶液在受控的温度水平（35℃）。 </li><li>当记录室的入口和出口被关闭，使用一个定制玻璃吸管滴管转移并放置展开海马到记录室中。在显微镜下，使用常规的小漆刷而组织处于漂浮于溶液定位展开海马。将展开与海马的alveus面朝下，CA3区背向和CA1场指向研究员。 </l我&gt; <li>使用真空吸管从记录室的边缘，以降低该溶液的水平，直至所述腔室进行干燥，所述组织被降低到阵列小心吸走在腔室中的溶液中。然后，小心地放置在组织上的一个顶部定制组织锚定（ 图4）（在具体的材料和设备第14号），以保持展开的海马到阵列上。放几滴溶液到记录室重新填充它，并逐渐打开入口和出口，以调节流动速率，在第IV滴注室每秒大约2滴。 </li><li>孵育录音室与正常脑脊液中的组织约1分钟，回收，然后切换所述溶液供应到4-AP溶于脑脊液和适当调整流速。孵育所述组织在4-AP溶于脑脊液为约5至10分钟，然后研究人员可以启动软件时，会出现自发性活动来记录信号。 </li></ol> 5.删除组织从PMEA的实验后<ol><li>控制的组织锚定由一个显微和逐步解除从记录室中的组织。关闭入口和出口停止录音室中的流动。录音室一应具全用溶液或加几滴解决方案，填补了腔，如果录音室没有满。 </li><li>用小画笔解除所述组织的各角。如果组织未漂浮在溶液中，然后使用真空管来与组织仍然坐在阵列上仔细干燥室中。然后小心地打开进，逐步填充室并关闭入口停止流动时的录音室已满。应用小漆刷再次抬起组织的每个角落。 </li><li>重复步骤5.2，直到组织被从阵列中分离出来并漂浮在溶液中。如果组织被漂浮在溶液中，然后用真空管吸组织消失。打开吨他流在入口和打开出口处的真空。用蒸馏水清洗系统和干出来。 </li></ol>

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The authors have nothing to disclose.

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?...

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。

<table><tbody><tr><td>desiccator jar</td><td>LABRECYCLERS Inc.</td><td>5410</td><td>Place regular paper towels at the bottome of the jar for animal anesthesia use.&amp;nbsp;</td></tr><tr><td>A blade and Custome made surgical stage for unfolding hippocampus</td><td>N/A</td><td>N/A</td><td>A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.&amp;nbsp;</td></tr><tr><td>Custom made tissue recovery chamber</td><td>N/A</td><td>N/A</td><td>Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.</td></tr><tr><td>Straight Operating&amp;nbsp;Scissors</td><td>Fisher Scientific</td><td>S17336B&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Medco Instruments No.:81995&amp;nbsp;</td><td>This scissors is used to&amp;nbsp;&amp;nbsp; decapitate the mice.</td></tr><tr><td>Integra Miltex Goldman-Fox&amp;nbsp;Scissors</td><td>Fisher Scientific</td><td>12-460-517&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; MILTEX INC&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; No.:5-SC-320</td><td>This scissors is used to cut the skull of the mice.&amp;nbsp;</td></tr><tr><td>Miltex&lt;br/&gt; Hysterectomy Forceps</td><td>Claflin Medical equipment</td><td>CESS-722033-00001</td><td>This Forceps is used to peel the cut skull to expose the brain</td></tr><tr><td>Micro Spatula</td><td>Cardinal Health</td><td></td><td>This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.&amp;nbsp;</td></tr><tr><td>Frey Scientific Stainless Steel Semi-Micro Spatula</td><td>Cardinal Health</td><td></td><td>this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.</td></tr><tr><td>small paint brush</td><td>Lowe&#039;s</td><td>tem #:&amp;nbsp;105657&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Model #:&amp;nbsp;90219</td><td>The one with the smallest size in a normal paint brush package</td></tr><tr><td>Fire polished glass help tool</td><td>N/A</td><td>N/A</td><td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td></tr><tr><td>Custom made glass needle</td><td>N/A</td><td>N/A</td><td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td></tr><tr><td>Custom made glass tool with a metal wire loop</td><td>N/A</td><td>N/A</td><td>This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.</td></tr><tr><td>Custom made glass solution dropper</td><td>N/A</td><td>N/A</td><td>This tool was&amp;nbsp; made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.</td></tr><tr><td>Custom made tissue anchor</td><td>N/A</td><td>N/A</td><td>Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.&amp;nbsp;</td></tr><tr><td>Custom fabricated microelectrode array</td><td>N/A</td><td>N/A</td><td>More detail about the array please refer to&amp;nbsp; Kibler, et al, 2011.&amp;nbsp;</td></tr><tr><td>Custom made filter and amplifiers circuits for the array</td><td>N/A</td><td>N/A</td><td>More detail about the array please refer to&amp;nbsp; Kibler, et al, 2011.&amp;nbsp;</td></tr><tr><td>Data acquisition processor 3400a</td><td>Microstar Laboratories</td><td>N/A</td><td>This is a complete data acquisition system with A/D converter.</td></tr></tbody></table>

neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array

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。

在这里图中所示的数据被记录在未折叠海马制剂与4-AP（100μM）脑脊液录音室中的组织的孵育期间在RT加入（25℃）。正常情况下活动开始不到5分钟，但在从旧有的动物海马组织中，可能需要更长的时间。与PMEA观察到的4-AP诱发的神经元放电是相同的如先前报道14,15。由于电极有200μm的高度，电极尖端刚好位于细胞层（ 图3C），因为电池层通常为250至300微米的海马alveus以上（ <str...

Watch this Scientific Journal Video about Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array at JoVE.com

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

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。

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

neural-activity-propagation-an-unfolded-hippocampal-preparation-with

Research

JoVE Journal

Neuroscience

8.3K 次观看.  Case Western Reserve University. 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。 

视频: Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

科研

神经科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

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 ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

医学

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

生物工程

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

<meta charset="utf8"></head><body>使用 CMOS-HD-MEA 一致地诱导和记录癫痫样活动

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures and ex vivo brain slices in unprecedented electrophysiological detail. CMOS-HD-MEAs were first optimized to record high-quality neuronal unit activity from cell cultures but have also been shown to produce quality data from acute retinal and cerebellar slices. Researchers have recently used CMOS-HD-MEAs to record local field potentials (LFPs) from acute, cortical rodent brain slices. One LFP of interest is seizure-like activity. While many users have produced brief, spontaneous epileptiform discharges using CMOS-HD-MEAs, few users reliably produce quality seizure-like activity. Many factors may contribute to this difficulty, including electrical noise, the inconsistent nature of producing seizure-like activity when using submerged recording chambers, and the limitation that 2D CMOS-MEA chips only record from the surface of the brain slice. The techniques detailed in this protocol should enable users to consistently induce and record high-quality seizure-like activity from acute brain slices with a CMOS-HD-MEA system. In addition, this protocol outlines the proper treatment of CMOS-HD-MEA chips, the management of solutions and brain slices during experimentation, and equipment maintenance.

<meta charset="utf8"></head><body>作者聚焦：使用 CMOS 高密度微电极阵列系统解开癫痫持续状态的癫痫发作动力学和新疗法

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain slices.

使用互补金属氧化物半导体高密度微电极阵列系统从急性脑切片中获得高质量的癫痫样活性

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

神经活动的传播的展开海马准备具有穿透微电极阵列

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使用互补金属氧化物半导体高密度微电极阵列系统从急性脑切片中获得高质量的癫痫样活性

使用互补金属氧化物半导体高密度微电极阵列系统从急性脑切片中获得高质量的癫痫样活性

使用互补金属氧化物半导体高密度微电极阵列系统从急性脑切片中获得高质量的癫痫样活性