Name: direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation
Uploaded: 2016-06-07T14:15:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

We are grateful for the technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was supported by the National Science Council (102-2320-B-001-026-MY3 and 100-2311-B-001-003-MY3) and Neuroscience Program of Academia Sinica.

涉及受试动物的程序是由机构动物护理和利用委员会，中央研究院，台北，台湾批准。 1.准备多电极阵列记录实验方案及设备<ol><li>制备人造脑脊髓液（ACSF; 124 mM氯化钠，4.4毫摩尔的KCl，1mM的的NaH 2 PO 3，2毫硫酸镁 ，2毫氯化钙 ，25mM的碳酸氢钠 ，和10mM葡萄糖，用95％氧气鼓泡2和5％的CO 2）。 </li><li>使用两种类型的MEA的探针:6×10平面MEA和8×8的MEA。前者探针盖，其包括皮质，纹状体和丘脑的区域。后者探头只包括皮质区域。 </li><li>使用60通道放大器0.1 Hz和1200扩增3千赫之间设置一个带通滤波器。收购在10 kHz采样率的数据。 </li><li>将两个氯化银涂层的银线的MEA室里面的DCS。使用氯化银涂覆的银线，以产生由一个孤立的刺激产生的电场。 </li><li>放置一个钨电极（直径，127微米;长度，7.62厘米; 8°的AC锥形尖端;电阻，5MΩ）为丘脑刺激，并放置在MEA室中的参比电极。交付使用由脉冲发生器控制的分离的激励器的钨电极的电流。 </li></ol> 2.脑切片准备<ol><li>使用雄性C57BL / 6J小鼠，4-8周龄。房子动物在空调房间（21-23℃，湿度50％; 12小时/ 12小时亮/暗循环，在8:00 am开灯），自由获取食物和水。 </li><li>把在步骤1.1中制备的脑脊液的250毫升等分试样，并将其放置在含有冰的烧杯中。与此同时，供给它由95％O 2和5％CO 2的连续气体。 </li><li>手术<ol><li>麻醉，用4％的异氟醚的动物中的玻璃框约3分钟。一旦动物达到手术的麻醉深度（由于缺乏脚趾掐响应的指示），将它放在一个浅盘一个充满碎冰，并用剪刀去除头。 </li><li>暴露颅骨，并剪掉剩余的肌肉。接下来，用咬骨钳，剥远离大脑头骨的背水面。剪掉用咬骨钳头骨的侧面。消毒所有手术器械的具有75％的乙醇溶液。 </li><li>用刮刀切嗅球和​​神经连接沿大脑腹面，并取出大脑。断头后，大脑迅速转移到填充有冰冷含氧学联的烧杯中。 </li></ol></li><li>内侧丘脑（MT）-ACC脑片的制备 注意:准备包含从MT到ACC 13通路切片。 <ol><li>手切两个削减矢状脑块2.0毫米外侧中线在每个半球显示皮层下解剖。然后提出两个斜切。使交叉切割平行于纹状体可见纤维束的第一。 </li><li>使从该中点的前连合和视束是腹侧和平行于thalamocingulate通路之间小​​脑和视觉皮层之间的连接的第二横切。 </li><li>附加脑块的角板（〜120°）与腈基丙烯酸酯粘合剂，和使切割正好途径的转折点的上​​方。展开板，压平，然后把它粘到vibratome的室内舞台。 </li><li>使内侧丘脑-ACC脑切片（500微米厚），然后沉浸其中在冰冷含氧aCSF.Transfer切片到录音室，而根据连续灌流（12毫升/分钟）保持在32℃下用含氧脑脊液1小时。 </li></ol></li></ol> 3.灌注腔的研制多电极阵列记录<ol><li>制备灌注腔<ol><li>放置一个多通道系统上的MEA的探针，并使用两个单独的聚乙烯管，以将探头连接到蠕动泵。用一个管引导脑脊液到MEA腔和其他管引导脑脊液出室。最后，不断灌注用温水（29-30℃）含氧学联（8毫升/分钟）的规定编制。 </li></ol></li><li>脑转移切片MEA。按住用湿棉签MEA大脑切片。仔细动脑子片，保证了ACC是面向在电极上方。 </li><li>使用切片锚套件和保持起伏按脑片。这个步骤保证了片和电极之间的良好的电连接。 </li></ol>通过DCS电场4代注意:在电场方向的定义是基于在ACC的axodendritic轴的方向。树突和索玛车厢的方向是使用高尔基染色12证实。 <ol><li>放置氯化银电极（定义为阳极）的近端到ACC，以及放置在另一电极（定义为阴极）远端到ACC。记录由所述两个场取向（平行和垂直于ACC axodendritic纤维）通过在MEA中产生的磁场强度，并提供使用刺激电场的电流。 </li><li>修复氯化银电极（约1.5-2厘米）的距离，并调整刺激的电流强度，使DCS的0.5和2 3mA之间。 </li></ol> 5.电致皮层突触反应注意:通过在MT电刺激，其中，一个可编程的电刺激发生器产生的矩形双相电流脉冲诱导在ACC突触反应。 <ol><li>重复上述第3节。 </li><li>放置在MT钨电极，并且从刺激器传送脉冲到贫通过双极钨电极片酰胺区域。 </li><li>使用不同的电流强度，以确定引起ACC的反应阈值。在这里，采用±150μA的强度和持续时间200微秒，这引起了ACC在大多数切片80％的最大反应。 </li><li>移动沿thalamocingulate途径（从MT能够胼胝体）的钨电极在MT-ACC切片，以获得最佳的响应曲线。 </li><li>使ACC对10-20扫描，并使用软件自动平均所有MT刺激诱发ACC的。 ISS在ACC由MT-ACC途径从MT刺激诱发突触反应的结果。 </li></ol> 6.电诱发癫痫样活动注意:癫痫样活性诱导的4-氨基吡啶的应用（4-AP; 250μM）和荷包牡丹碱（5μM）。上一个时间控制的研究表明，最大的和稳定的反应出现药后应用14 2-3小时。 <ol><li>重复上述第5节。 </li><li>添加药物的灌注溶液中。使用4-AP（250μM）和荷包牡丹碱（5μM）。混合均匀的药物，继续灌注2-3小时。 </li><li>为了便于癫痫样活动，保持在一个相对较快的灌注速率（8毫升/分钟），这也可以帮助防止pH梯度的积聚灌注泵。 </li><li>放置在一个MT钨极，并提供电刺激（150μA，200微秒持续时间），以获得ACC响应曲线。 </li><li>让10-20扫描和平均响应。 </li><li>替换用新鲜脑脊液灌注溶液洗出药物。重复步骤6.5。 </li></ol> DCS对诱发皮层响应7.测试效果<ol><li>并重复第3 4.确保均匀电场通过使被置于其内M个两个平行的AgCl涂层的银导线之间的电流产生的EA室。如果没有问题，在DCS停留0.5和2 3mA之间。 </li><li>关闭DCS，并把钨电极刺激丘脑（±150μA，200微秒持续时间）。为了获得在ACC最大突触反应，使10-20扫描和平均响应。 </li><li>同时打开的DCS（2毫伏/毫米DCS强度）和丘脑刺激（350微安，200微秒持续时间）。评估DCS在丘脑刺激诱发的ACC反应的幅度的变化。 </li><li>关掉DCS和添加4-AP（250μM）和荷包牡丹碱（5微米），以灌注溶液。然后等待2-3个小时。当药物影响大脑切片，切片生产皮质发作的反应。 </li><li>使ACC对10-20扫描，然后测量电诱发癫痫发作皮质反应的幅度和持续时间。 </li><li>步骤7.5之后，同时打开在DCS（2毫伏/毫米DCS强度）和丘脑刺激（150微安，200 durati在微秒）。在评估过程中DCS应用的幅度和皮质诱发癫痫发作的反应持续时间的变化。 </li><li>更换新学联灌注液洗出的药物，并重复步骤7.2和7.3。 </li><li>收集所有的记录数据，并且数据分组为不同的实验条件。评估不同的实验条件下，皮质癫痫响应的幅度和持续时间。 </li></ol> 8.数据分析<ol><li>使用软件（ 如 MC机架软件）来自动平均记录的响应，以及原始数据导出到电子表格。分析原始数据的幅度和持续时间，并产生颜色的数字。 </li><li>以检测振荡发作的事件，使用软件来测量基准值和标准偏差（SD）。设置3 SD噪声电平阈值。振荡活动期间的峰值振幅是超过这个阈值的自动检测编辑。 </li><li>执行使用学生的t检验进行统计分析。 </li><li>明示的测量和在文本以平均值±SE的方差（ANOVA）结果单向分析，其中n指示的片的数目的研究12。 </li></ol>

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The authors declare that they have no competing interests.

在本研究中，DCS系统上ACC癫痫样活动的持续时间和方向的影响进行了试验。以获得在小鼠脑切片稳定的数据，如何保持在MT-ACC通路的完整性，并避免损坏它是关键，特别是在其中两个成角度的腹侧削减和皮质的背切制成的步骤。此外，以制备脑切片的时间也可以影响脑切片，这应该是尽可能短的时间，以保持大脑新鲜和强的活性。先前的研究表明，对靶组织的电化学损伤可在体内制备15发生。 体外脑切片制剂可用于避免这一问题。 在体外制备中，组织不直接接触的电极，从而最大限度地减少电化学效应16。 DCS的影响与被定向在不同的方向上该电极进行了比较。当该电极ES在90°和270°取向，DCS没有影响诱发活性（ 图3）。因此，这种对照实验排除了电化学反应的任何副作用的损坏在我们的研究组织DCS的可能性。大脑切片的保护恢复方法是另一个关键;在这项研究中的脑脊液公式提供到保护切割方法的替代，并且是在脑切片的神经元保存从4至8周龄的动物的高度有效的。该方法不适合所有年龄的动物中使用;使用的Tris脑脊液的出现是在老年小鼠6周，老年人使用NMDG保护回收方法是有效的年轻小鼠。因此，用户应该牢记的相对年龄换算公式跨越物种选择实验的最佳方法。 使用的MEA记录大脑切片中的常用技术，但一个电场与MEA记录SYS组合统通常不进行。影响到MEA记录系统的半导体解决方案的直流场是一个有趣的方法，特别是对于以分钟多少​​秒时间。先前的研究表明，DCS应用没有改变pH值在脑脊液溶液，这表明该导电溶液的pH为在该实验装置12稳定。相对快速灌注率（8毫升/分钟）保持以促进癫痫样活动，并在MEA探针的化学变化的任何产物进行灌注冲洗掉，从而避免了pH梯度的积聚。多电极阵列记录技术往往是由脑切片和范围记录电极的类型的限制。类型脑切片的确定哪个电路通路被记录下来，并且该记录电极的范围内确定的单个或多个脑核是否被记录。这些条件必须在实验之前进行确认。 预<html> vious研究表明，DCS的长期影响通过突触传递17的调制发生。在本研究中，阴极的DCS在MT-ACC途径引起LTD。所述LTD。或发作相关增强的depotentiation提议是癫痫发作抑制的潜在机制的一部分，这表明TDCS治疗的结果的该增强是可能的。然而，没有发表的研究一直专注于在扣带皮层的场强。在皮层的内侧部分的扣带皮层的深位置难以测试。例如，这是不可避免的电流流动可能影响是更接近表面的组织和血管。通过TDCS针对深部组织的难度可能会限制TDCS 体内研究中的应用。因此，要理解的DCS是如何影响神经元活动，一个脑切片准备应被使用，如非特异性血管效应需要排除。 jove_content"&gt;对于建立一个实验模型的目的，所描述的查封是在一个健康的大脑引起的。在癫痫样活动是由一个电脉冲进一步诱发。在癫痫发作的时间可以精确控制被应用DCS时该结果可为TDCS治疗的更多信息。另一个重要发现是长期在被诱导TDCS区域皮质兴奋性的变化。在未来，如果TDCS的基本机制可以阐明，那么DCS的组合，药物治疗以增强LTD。在治疗癫痫症可能是一个非常有趣的发展。 总之，提供了调查DCS对thalamocingulate和胼突触可塑性和急性发作的影响的方法。 DCS对我们的大脑切片准备癫痫样活性的长期影响通过LTD样的机制发生。

Epilepsy is a common neurological disorder. Thirty percent of patients with epilepsy suffer from drug-resistant seizures1. Transcranial direct-current stimulation (tDCS) provides a noninvasive approach to control or alter network activities across large brain areas, such as seizures. Clinical studies have shown that tDCS effectively treats intractable seizures2 and can produce both short- and long-term suppressive effects on seizures3-5. However, the therapeutic mechanism of tDCS actions is still unclear. The brain slice model presented is an in vitro method to investigate how the therapeutic mechanism of tDCS actions alters the symptoms of seizure-like brain activities. Accordingly, to achieve its optimal effects, specific stimulation parameters including orientation, field strength, and stimulation duration need to be tested in an experimental model. Previous studies have shown that the orientation of the electric field is important to obtain therapeutic effects6. Thus, testing and arranging the orientation of electrodes relative to the position of the tested brain slice are feasible.
Frontal lobe epilepsy and anterior cingulate cortex (ACC) seizures are often drug-resistant7,8. Some studies have reported the application of tDCS in the cingulate cortex9-11. tDCS is shown to affect vigilance, decision making and emotion through alteration of ACC activities, and can modulate neuronal excitability and seizure activity in this brain region12. Therefore, suppressive effects of tDCS on ACC seizures might be helpful for clinical treatment and the evaluation of alternative treatments.
The present protocol describes the preparation of an electrode in the recording chamber for DCS of a brain slice and its effect on seizure-like activity recording with a multielectrode array (MEA).

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;aCSF (total:1 L):&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D(+)-Glucose</td><td>MERCK</td><td>1.08337.1000</td><td>10 mM</td></tr><tr><td>Sodium hydrogen carbonate</td><td>MERCK</td><td>1.06329.0500</td><td>25 mM</td></tr><tr><td>Sodium chloride</td><td>MERCK</td><td>1.06404.1000</td><td>124 mM</td></tr><tr><td>(+)-Sodium L-ascorbate, &gt;=98%</td><td>SIGMA</td><td>A4034-100G</td><td>0.15 g/2 c.c</td></tr><tr><td>Magnesium sulfate, anhydrous, ReagentPlus</td><td>SIGMA</td><td>M7506-500G</td><td>2 mM</td></tr><tr><td>Calcium chloride dihydrate</td><td>MERCK</td><td>1.02382.1000</td><td>2 mM</td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>MERCK</td><td>1.06346.1000</td><td>1 mM</td></tr><tr><td>Potassium chloride</td><td>May &amp;amp; Baker LTD Dagenham England</td><td>MS 7616</td><td>4.4 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(+)-Bicuculline</td><td>TOCRIS</td><td>0130</td><td>5 &amp;micro;M in aCSF</td></tr><tr><td>4-Aminopyridine</td><td>TOCRIS</td><td>0940</td><td>250 &amp;micro;M in aCSF</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Brain slice Preparation:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vibratome</td><td>Vibratome</td><td>Series 1000</td><td>Block slicing into 500 &amp;micro;m thick slices</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;MEA system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multielectrode array (MEA) probes: 6 x 10 planar MEA</td><td>Multi Channel Systems</td><td>60MEA500/30iR-Ti-pr MEAS 6x10</td><td>electrode diameter, 30 &amp;micro;m; electrode spacing, 500 &amp;micro;m; impedance, 50 k&amp;Omega; at 200 Hz</td></tr><tr><td>Multielectrode array (MEA) probes: 8 x 8 MEA&amp;nbsp;</td><td>Ayanda Biosystems</td><td>60MEA200/10iR-Ti-pr MEAS 8x8</td><td>pyramidal-shaped electrode; diameter, 40 &amp;micro;m; tip height, 50 &amp;micro;m; electrode spacing, 200 &amp;micro;m; impedance, 1,000 k&amp;Omega; at 200 Hz</td></tr><tr><td>A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1,200X amplification</td><td>Multi-Channel Systems</td><td>MEA-1060-BC</td><td></td></tr><tr><td>MC Rack software at a 10 KHz sampling rate</td><td>Multi-Channel Systems</td><td></td><td>Software for data collect and recordings</td></tr><tr><td>control of a pulse generator</td><td>Multi-Channel Systems</td><td>STG 1002</td><td></td></tr><tr><td>slice anchor kits and hold-downs</td><td>Warner Instruments</td><td>SHD-26H/10; WI64-0250</td><td></td></tr><tr><td>Peristaltic Pump-minipuls3</td><td>Gilsom</td><td>MINIPULS3</td><td>perfusion rate : 8 ml/min</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Stimulation system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isolated stimulator</td><td>A-M Systems</td><td>Model 2100</td><td>intensity of &amp;plusmn;350 &amp;mu;A , duration of 200 &amp;mu;sec</td></tr><tr><td>Tungsten electrode</td><td>A-M Systems</td><td>575300</td><td>placed in thalamus</td></tr></tbody></table>

direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation

阴极经颅直流电刺激（TDCS）诱导的耐药性癫痫发作的抑制作用。要进行有效的行动，刺激参数（ 例如 ，方向，电场强度和持续时间的刺激），需要在老鼠大脑切片筹备工作进行检查。测试和安排相对于小鼠脑切片的位置的电极的取向是可行的。本方法保留thalamocingulate途径来评估DCS对前扣带皮层癫痫样活动的影响。多信道阵列记录的结果表明，阴极DCS显著降低的刺激诱发反应的4-氨基吡啶和荷包牡丹碱诱导的癫痫样活动的振幅和持续时间。这项研究还发现，在15分钟阴极DCS应用程序引起的长期萧条的thalamocingulate途径。本研究探讨DCS对thalamocingulat的影响Ë突触可塑性和急性发作之类的活动。当前程序可以测试最佳刺激参数，包括方向，磁场强度，并在体外的小鼠模型刺激持续时间。此外，该方法可以评估的DCS上皮层癫痫样活动在细胞和网络两级的作用。

在Thalamocingulate切片和MEA记录系统设置的准备从小鼠MT-ACC切片是一种特殊的切片准备，允许thalamocingulate通路的电生理特性的探索。 图1A示出了制备MT-ACC切片的方式。老鼠的大脑迅速取出并存放于阴凉含氧学联（ 图1A，A，B）。揭示皮层下解剖，将脑切2.0毫米横向从中线在每个半球获得矢状脑块（ 图1A，C，D）。两个成角度的腹侧削减了在大脑中的块作出保留thalamocingulate通路。第一横切口是在大脑块的前面制成，平行于在纹状体中可见纤维束。第二横切于脑块从连接下注后制成吐温小脑和视觉皮层的前连合和视束之间的中点。切口作了后，大脑块被粘在成角度的塑料板（120°角），和一个切直接通过皮质（ 图1A，例如 ）在背侧制成。大脑块板粘在vibratome和凉爽的含氧学联淹没。最后，几片（500微米厚）被从脑块中取出，并氧化学联孵育（ 图1A，H，I）。 图1B示出在MEA记录系统的设置。的示意图示出了连接到记录计算机的灌注和MEA系统。空MEA探针置于放大器​​内部，并灌注开始以8毫升/分钟的流速。在MT-ACC脑切片放置在MEA探针同时确保的记录区域是尽可能靠近于中心。刺激剂是使用d，来刺激脑切片并产生电场。所有的步骤完成后，该系统记录的thalamocingulate通路的电生理特性。 图1C示出的记录区域图和在其中的MT-ACC脑切片放置在MEA探头的方式。 图1C-a显示在MEA探针的外观。探头上的黑线（红色箭头）帮助用户确定所述放大器内的探头的正确方向。 图1C-b显示在MEA探针的主要电路。 图1C-C示出了在覆盖在电阵列皮层组织。 测试诱发反应为了确认在切片准备thalamocingulate途径保存，这项研究刺激丘脑并在电生理实验在ACC记录。仅当在MT传送的少量电流是在实验中使用与在ACC突触后电位切片。 图2A示出在ACC的刺激和记录的电极和典型丘脑刺激诱 ​​发反应的位置。为了诱导癫痫样活动，4-AP（250μM）和荷包牡丹碱（5μM）被用来诱导癫痫样活动。典型的4-AP用荷包牡丹碱诱导的自发癫痫样活性组成的发作的发病，随后是强直期持续时间长。被选定为在图2B的放大倍率的痕迹。这项研究还试图诱导药物引起癫痫发作后，在MT递送刺激。 4-氨基吡啶/荷包牡丹碱诱发癫痫样活动被电刺激（ 图2C）后诱导。 测试DCS和切片的方向先前的临床研究表明，阴极的DCS的电场的取向影响丘脑刺激诱 ​​发活性。 图3A示出电场，它被安排平行或垂直于在ACC枝晶和胞体室的取向不同的取向。当电场被布置成平行于神经元细胞，阴极刺激抑制在ACC（ 图3B的内侧部分丘脑刺激诱 ​​发反应， 上图）。当电场被布置成垂直于神经元细胞，观察到（ 图3B，下图）上丘脑刺激诱 ​​发反应无显著影响。并行阴极DCS也能抑制4-AP-并在ACC（ 图3C，上图）的内侧部分荷包牡丹碱诱导的癫痫样的活动。它缩短癫痫样活动的持续时间，并观察到（ 图3C，下图）垂直阴极的DCS的无显著效果。这些结果证实，电场的方向是在thalamocingulate通路调控突触传递重要。 DCS对癫痫发作影响经颅磁刺激的临床应用，TDCS和DCS提供了耐药性发作的治疗无创的方法。以往的研究表明，场刺激调​​制各脑区突触可塑性的影响癫痫样活动。本结果表明，阴极TDCS压下thalamocingulate突触传递。刺激诱 ​​发反应和癫痫样活动的持续时间的幅度被按下（11 9的片，81.82％; 图URE 4A，左图）。 图4A（右图）显示，阴极DCS的15分钟有效地诱导长期抑郁（LTD）在MT-ACC途径和抑郁诱发反应（N = 11，P &lt;0.05）。 图4B（左图）显示，丘脑刺激在扣带皮层诱发健壮癫痫样活性。经过三十分钟15分钟阴极DCS应用，丘脑刺激诱发癫痫样活动的持续时间缩短。研究结果还表明，癫痫发作持续时间15分钟后，阴极DCS没有DCS应用（N = 9，P &lt;0.05相比显著下降; 图4B， 右图）。 <img alt="图1" src="/files/ftp_upload/53709/53709fig1.jpg" /> 图1:在Thalamocingulate切片和MEA记录系统设置的准备 （ 一 ）MT-ACC切片亲cedure（ 一 ）消除大脑和（b）转移到冷却含氧学联。 （ 三 ）建立从中线2矢状窦旁切口。脑块的内侧部分（ 四 ）侧视图。 （ 五 ）使大脑块的两个成角度腹削减。 （F）的胶块大脑到一个角度的塑料板。 （g）在背部切口，展开脑块。 （H）胶在vibratome脑块。 （ⅰ）收集从大脑块切片。 （B）MEA记录系统设置。 （C）的MEA记录区域。 （ 一 ）MEA探头。 （ 二 ）MEA电路。 （ 三 ）脑片的ACC电极探针上面导向。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53709/53709fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> E ="1"&gt; <img alt="图2" src="/files/ftp_upload/53709/53709fig2.jpg" /> 图2:不同的诱发反应丘脑刺激和药物引起的刺激在ACC（A）丘脑刺激诱 ​​发反应。 （B）4 Aminopyridine-和荷包牡丹碱诱导的癫痫样的活动。 （C）丘脑stimulation-和药物引起的癫痫样的活动。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53709/53709fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53709/53709fig3.jpg" /> 图3:DCS的不同方向的影响 （ 一 ）电场的不同方向。 （B）中与DCS丘脑刺激诱 ​​发反应。阴极DCS（ 三 ）对癫痫样的活动。://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53709/53709fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53709/53709fig4.jpg" /> 图4:阴极TDCS对癫痫发作 （A）十五分钟阴极DCS诱导LTD郁闷诱发性的影响 。 （B）的癫痫样活动的发生阴极刺激期间下降。癫痫样活性的15分钟阴极DCS即使DCS的应用被终止忍受的抑制。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53709/53709fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

direct-current-stimulation-multi-electrode-array-recording-seizure

Universidad San Sebastian

Research

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Neuroscience

10.4K 次观看.  Academia Sinica. Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

视频: Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

科研

神经科学

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

经颅直接电流刺激电极定位和蒙太奇

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

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

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) ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

啮齿类动物和人体组织急性 ictal 活性的产生及按需启动

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

<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 ...

Monitoring Seizure-Induced Neural Electrical Activity in Brain Slices Using Microelectrode Arrays

Source: Panuccio, G., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57548">Recording and modulation of epileptiform activity in rodent brain slices coupled to microelectrode arrays.</a> J. Vis. Exp. (2018)
This protocol involves using a microelectrode array (MEA) to record electrical activity in a brain slice exposed to a seizure-inducing drug. The procedure includes positioning the brain slice in an MEA recording chamber, applying electrical stimulation to induce artificial seizures, and analyzing the resulting signals.

使用微电极阵列监测脑切片中癫痫发作诱导的神经电活动

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of the MEA ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Impact of Direct Current Stimulation on Evoked Epileptic Seizures in Mouse Brain Slices

Source: Lu, H., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53709">Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation.</a> J. Vis. Exp. (2016)
The video demonstrates a study for assessing the impact of direct current stimulation (DCS) on epileptic seizures in mouse brain slices. The brain slices containing the connected medial thalamus (MT) and anterior cingulate cortex (ACC) regions are placed in a multielectrode array (MEA). Electrical stimuli to the MT and seizure-inducing drugs are applied to induce epileptic seizures in the ACC. DCS is then applied to assess its impact on evoked seizures.

直流电刺激对小鼠脑切片中诱发性癫痫发作的影响

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing Experimental Solution ...

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

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此视频中的章节

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

经颅直接电流刺激电极定位和蒙太奇

人类癫痫术后皮层组织的多电极阵列录音

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

啮齿类动物和人体组织急性 ictal 活性的产生及按需启动

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

使用微电极阵列监测脑切片中癫痫发作诱导的神经电活动

直流电刺激对小鼠脑切片中诱发性癫痫发作的影响