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 border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1017px;">Anesthetic:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td style="width:187px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">aCSF (total:1L):</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td style="width:187px;">10 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td style="width:187px;">25 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td style="width:187px;">124 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(+)-Sodium L-ascorbate, &#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td style="width:187px;">0.15 g / 2 c.c</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate, anhydrous,ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td style="width:187px;">1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td style="width:187px;">4.4 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Drugs:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Bicuculline</td>
			<td>TOCRIS</td>
			<td>0130</td>
			<td style="width:187px;">5 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Aminopyridine</td>
			<td>TOCRIS</td>
			<td>0940</td>
			<td style="width:187px;">250 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Brain slice Preparation:</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Vibratome</td>
			<td>Vibratome</td>
			<td>Series 1000</td>
			<td style="width:187px;">Block slicing into 500 &#181;m thick slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">MEA system:</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:355px;">Multielectrode array (MEA) probes: 6 x 10 planar MEA</td>
			<td>Multi Channel Systems</td>
			<td style="width:184px;">60MEA500/30iR-Ti-pr MEAS 6x10</td>
			<td style="width:187px;">electrode diameter, 30 &#181;m; electrode spacing, 500 &#181;m; impedance, 50 k&#937; at 200 Hz</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:355px;">Multielectrode array (MEA) probes: 8 x 8 MEA&#160;</td>
			<td>Ayanda Biosystems</td>
			<td style="width:184px;">60MEA200/10iR-Ti-pr MEAS 8x8</td>
			<td style="width:187px;">pyramidal-shaped electrode; diameter, 40 &#181;m; tip height, 50 &#181;m; electrode spacing, 200 &#181;m; impedance, 1000 k&#937; at 200 Hz</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:355px;">A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1200X amplification</td>
			<td>Multi-Channel Systems</td>
			<td>MEA-1060-BC</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MC Rack software at a 10 KHz sampling rate</td>
			<td>Multi-Channel Systems</td>
			<td></td>
			<td style="width:187px;">Software for data collect and recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">control of a pulse generator</td>
			<td>Multi-Channel Systems</td>
			<td>STG 1002</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">slice anchor kits and hold-downs</td>
			<td>Warner Instruments</td>
			<td style="width:184px;">SHD-26H/10; WI64-0250</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump-minipuls3</td>
			<td>Gilsom</td>
			<td style="width:184px;">MINIPULS3</td>
			<td>perfusion rate : 8 ml/min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Stimulation system:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isolated stimulator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td style="width:187px;">intensity of &#177;350 &#956;A , duration of 200 &#956;s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten electrode</td>
			<td>A-M Systems</td>
			<td>575300</td>
			<td style="width:187px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

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 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 ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

在分析癫痫样活动和运动的一种低成本方法<em&gt;果蝇</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

设计和超轻重量的制作，可调节的多电极电生理记录小鼠探头

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

经颅直流电刺激 (tDCS) 在小鼠中的作用

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

使用具有高清晰度颅内直接电流刺激的 3D 数字化仪确定刺激位置

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.