他是由欧洲联盟 MSCA-IF-2014 项目资助的玛丽居里夫人-居里研究员. b. 美国, g.a.n. 660689, 框架计划 H2020。GUI mapMEA 是免费提供的要求, 作者 ilaria.colombi@iit.it ilaria. colombi@iit。

<ol>
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作者没有什么可透露的。

多边环境协定是神经生理学调查的宝贵工具, 近年来由于勘探和发展已达到成熟。与常规场电位记录相比, 多边环境协定提供了较高的观测点和已知电极间距离的优势, 这对于准确定位神经元网络的相互作用至关重要。
该技术还可以结合其他电生理学方法, 如膜片钳记录15, 以研究单神经元和神经网络活动之间的关系。同时, 可视化场电位和多单位活动的可能性可以为小神经元群的活动与集体神经网络的相关性提供宝贵的见解。与电压敏感染料、钙成像和光遗传学29的结合允许使用多面体方法推断神经生理学现象.除了药理学研究, 在同一系统中进行记录和刺激的可能性使得该技术的记录技巧非常强大和多才多艺: 例如, 有可能研究突触可塑性现象在内存形成的核心30, 使用此处介绍的调节协议, 与 DBS 治疗癫痫和多种脑部疾病有关。最近的证据表明, 多边环境协定记录技术可以成功地应用于人类癫痫脑组织16表明它在癫痫研究中的宝贵用处, 既了解这种破坏性疾病的基本机制并微调 DBS 算法来改善它。
然而, 由于水下记录室的要求, 以及让大脑切片在微电极的固体基底上休息的必要性, 多边环境协定迫使对脑切片的 "极限" 条件下的电生理学实验进行追求。是集成的。因此, 脑组织可能无法获得足够的氧气供应, 这反过来又可能影响录音的质量。
这里描述的协议允许可靠地记录和研究 ictogenesis 在鼠边缘网络上的耦合, 利用急性体外4AP 模型和追求长期的电刺激, 为评估提供相关信息星展的政策。
以前对癫痫边缘网络的电调节的研究基于传统的场电位记录, 使用了老鼠或鼠脑切片, 结果类似24,25;但是, 由于与小鼠7获得的脑组织相比, 大鼠脑组织的使用被证明更具挑战性。关于多边环境协定技术, 小鼠脑切片最适合的优点是它们的体积较小。此外, 鉴于小鼠与大鼠脑组织中发作放电的发生率较高, 在一个实验日进行大量的脑切片测试是可能的, 这反过来又使数据收集速度更快、效率更高, 并能减少使用的动物数量。
对于现有方法的意义:
使用此处描述的自定义分庭记录的发作排放似乎与使用传统的多边环境协定环室和相同的多边环境协定类型 (平面微电极cf 17) 所观察到的持续时间和发生率相似。然而, 需要强调的是, 当使用常规的圆形记录室和低灌注率 (1 毫升/分钟) 时, 脑切片厚度必须显著降低, 这可能会阻碍调节实验的成功追求, 因为连接不良。
在本协议中, 由膜片钳记录室设计启发的低容量自定义记录室提供了稳定和可靠的层流, 这对于成功地追求多边环境协定录音至关重要;它还允许增加脑切片厚度为400µm, 以实现公平权衡组织的生存能力和内在的连通性。确实认识到, 腔内记录解决方案的层流是非常可取的脑切片电生理学, 因为它不受温度, 氧气和 pH 梯度的影响, 在循环流圆记录观察钱伯斯19,20,31 (像提供了商业上可用的多边环境协定)。这种梯度引入实验性偏倚, 也对脑切片有害。相对较小体积 (~ 1.5 毫升) 的记录室连同高灌注率 (5-6 毫升/分)16,31允许充分交换灌注介质 (≥3时间/分钟)。定制室可以很容易地获得从商业来源以实惠的价格或生产内部使用3D 印刷技术。其他研究报告了从400µm 厚的人脑切片16使用传统的多边环境协定环室的录音, 同时保持 ACSF 体积为1毫升, 并在低噪声蠕动泵的帮助下执行高灌注率 (5-6 毫升/分钟)。但是, 作者使用了不同的 ictogenesis 模型即, 低镁2 +, 这可能比4AP 模型的影响较小, ephaptic 机制涉及显著增加细胞外 K+浓度11,22. 我们发现, 4AP 模型中不需要高的灌注率, 可能是由于累积的胞外 K+的快速冲洗, 在录制 ACSF16中可能要显著增加。事实上, 当 ASCF 灌注速度增加到 2-3 毫升/分钟, 而脑切片被厚 ACSF 层淹没时, 发作事件出现了更多的 "块", 而在返回到较低的灌注率 (1 毫升/分钟) 和较薄的 ACSF 层在组织表面水平 (没有显示数据)。本协议中描述的自定义记录室允许以1毫升/分钟的流速交换灌注介质 3-5 次/分钟。因此, 即使在相对较低的灌注率下, 脑切片的整体氧气供应也得到了强烈的改善, 同时仍然保证了记录温度和高信噪比的稳定。最重要的是, 可以将胞外 K+浓度保持为生理值。
ictogenesis 的4AP 模型不需要对 ACSF 离子成分进行任何重大修改, 例如降低 Mg2 +或增加 K+, 并提供了保持兴奋和抑制传输完好的独特优势。12, 鉴于谷氨酸和 GABAergic 网络在癫痫发作同步中的关键作用 (cf 7), 这一方面与癫癎的研究高度相关。本协议中使用的 4 ap ACSF 包含生理 K+浓度 (3.25 毫米) 和略低的镁2 +浓度比持有 ACSF (1 毫米与1.3 毫米)。这种浓度仍然是在报告的镁2 +浓度在啮齿动物脑脊液中的生理价值, 并在许多实验室使用 (见例如17,18)。对于所述协议的目的, 我们发现, 这种轻微的减少是首选的援助, 以4AP 的效果。
在以前的工作中, 4AP 已被应用于脑切片, 当它已经定位在录音室17,18中。除非实验者需要观察4AP 应用与癫痫发作模式开始之间的时间滞后, 我们发现这种方法相当耗时, 不宜进行长时间的录音和刺激疗程。本议定书所述。32摄氏度的脑切片预孵化可节省大量实验时间, 因为脑切片可以在系列中预先处理, 同时使用其他组织切片进行实验。
脑切片的长期生存能力可能有助于分析长期的网络特征。此外, 如果实验者想要比较几种刺激方案, 这些方法必须在同一脑切片中进行, 以提高对重复电刺激的适应性, 这是很有好处的。在我们的手中, 当测试3刺激协议, 每一个之前的控制阶段, 然后是一个恢复阶段, 实验可能会持续 3-5 小时。在这种情况下, 实时制图是至关重要的, 以激活正确的途径和压制, 而不是青睐 ictogenesis 通过电刺激。我们的用户友好的 GUI 代表了一个快速, 简单和灵活的工具, 绘制不同的大脑结构的电极。与商业软件相反, 即使具有基本编程技能, 也可以添加自定义的多边环境协定布局。图像可以在明亮的领域获得;因此, 任何具有良好分辨率和适合显微镜的通用相机是合适的。倒置显微镜是必不可少的可视化的电极位置相对于脑切片, 因为这些将隐藏在组织下方, 如果使用直立显微镜。然而, 如果实验者需要进行刀切来扰乱特定的神经通路, 那么除了倒置型外, 还建议使用立体显微镜。
最后, 建议的方法的一个进一步好处是便宜和相对地容易装配多数必要的工具, 象自定义录音室和存放室、温暖的浴和切片锚, 以及不必要的使用昂贵的低噪音蠕动泵。
技术的局限性:
该记录不允许可视化非常慢的波浪,即, 信号中的 DC 移位。这种基线偏差可能有助于发作放电持续时间的渐进测量, 最重要的是, 它们是研究皮质扩张性抑郁症的基础 (一种与癫痫突发意外死亡有关的现象32 , 并共享癫痫与偏头痛之间33)。
关于电子调节, 这里所描述的协议允许在不影响脑切片生存能力的情况下进行几次刺激疗程。我们可以成功地执行多达3次的刺激会话, 20-45 分钟, 类似于以前工作中报告的25。虽然脑切片可能会经受更多的刺激疗程或更长的刺激协议, 但我们没有在这方面测试脑切片。我们建议将刺激方案的数量限制在 3, 避免延长 (≥60分钟) 刺激疗程, 这可能会显著地强调在这些限制条件下维持的脑组织, 但在刺激撤退时缺乏恢复。
议定书内的关键步骤:
几个关键因素可能会阻碍成功地追求记录和调制的癫痫活动的脑切片耦合到多边环境协定。除了所使用的啮齿动物种类和脑切片的质量, 录音过程中的灌注率和 ACSF 流的动态 (i. e, 层流与圆形) 在记录室内, 我们发现恢复的条件和脑切片的长期维护以及4AP 应用的模式是最关键的步骤。
在使用浸没式保温室时, 在室温下贮存脑切片, 以保持脑组织网络活动, 有利于4AP 应用发作放电的诱导, 是至关重要的。在我们的手, 恢复和维护在32°c 恶化了脑组织在 3-4 小时内切片。
然而, 4 ACSF 的脑切片在室温下孵化并记录在32摄氏度是不可取的。在这种情况下, 我们发现有许多困难在观察健壮的复发发作放电。事实上, 在20–24°c 范围内的低温已经报告, 以抑制或甚至防止 4 ap 诱发 ictogenesis 两个在体外34和在体内35。因此, 4AP 孵化和记录温度必须在 30-34 摄氏度以内, 必须匹配。为了避免把大脑组织置于太大的压力下, 由于同时诱导 hyperexcitability 4AP 和脑切片的突然暴露从室温到温暖 (32 °c) 4 ap-ACSF, 这是基本的执行中间预热步骤, 让大脑切片习惯在持有 ACSF 在32°c 20-30 分钟. 跳过此步骤可能会影响 ictogenesis 感应4AP。
另一个需要提及的方面是 ACSF 后的 d-葡萄糖浓度在切片后的重要性。25毫米的浓度保持脑切片优于10毫米浓度在许多实验室使用, 它也提高了发作活动的发生率, 这是2倍更快 (因此, 使其更容易进行长系列的实验规程在几脑切片每天)。
最后, 需要指出切片过程中的一些重要细节。首先, 不要让完整的大脑和脑切片冻结在冷切割 ACSF。为 < 2 分钟冷却的大脑是足够的, 因为小鼠的大脑大小。组织切片应立即从缓冲托盘转移到冲洗烧杯室温。冲洗切割 ACSF 是非常重要的, 否则高蔗糖浓度将使脑切片坚持在尼龙网眼的保持室。为了有效地冲洗组织, 重要的是尽量减少切割 ACSF 的内容内的转移吸管, 以避免污染的持有 ACSF 过度。2级冲洗有助于减少污染。切片程序完成后, 应立即将组织切片从冲洗烧杯转移到保温室, 在保持 ACSF 中, 软尼龙网暂停中途, 可使两侧的组织氧合。在切片过程后的所有阶段都应采用同样的原理: 脑切片不应该坐在烧杯的底部, 他们不应该接触到持有商会的两侧, 他们不应该接触对方, 也不应该重叠。
修改和疑难解答:
ACSF 成分可根据实验者的需要进行修改。例如, 可以添加药物来解剖特定的神经递质或离子通道对电调节的功效的贡献。此外, 可以省略丙酮和抗坏血酸 (cf 表 2), 尽管我们发现它们发挥了强大的神经保护作用。我们在表 3中报告了在本协议中可以遇到的最常见问题以及如何处理它们。
结论:
这一记录无疑是解决神经网络在健康和疾病中相互作用的宝贵技术。除了药理学研究, 还可以评估与星展集团应用于癫痫和其他神经系统疾病相关的电调节协议。在本议定书中, 我们已经表明, 有可能重现典型的周期性刺激实验, 其结果与常规细胞外磁场电位记录和外部刺激电极获得的效果相似。越来越多的商用用户友好设备和先进的软件工具, 使多边环境协定记录技术也适用于闭环刺激实验, 对脑组织提供特别刺激, 并研究反馈机制对神经网络反应的贡献。

癫痫是一种危及生命的渐进性紊乱, 导致大脑的不受控制的活动1;它携带着最高的疾病负担和重要的社会耻辱2,3。这是最常见的综合征 (40%) 和最频繁 (~ 30%) 抗癫痫药物治疗4。虽然癫痫组织的手术消融可以改善病人的病情, 但在所有患者中都是行不通的, 可能不能保证完全无癫痫的生活5。在药物治疗或神经外科不合适的情况下, 电星展集团对癫痫边缘网络的调制是一种很有前途的方法。
啮齿动物脑切片是研究神经元网络如何在健康和疾病中发挥作用的重要工具6通过体外电生理学技术, 因为它们保存, 至少部分, 大脑的原始结构和连通性感兴趣的区域 (ROI)。特别是, 水平结合的海马-嗅皮质 (海马-EC) 切片构成了所涉及的必要的神经网络, 因此在体外研究中经常使用7。
通过改变人工脑脊液 (ACSF) 的离子组成, 例如降低镁, 同时增加钾8,9,10或通过药理操作, 例如阻止抑制 GABAergic 活动 (参见11进行全面审查)。然而, 这些模型是基于激励和抑制的不平衡变化;因此, 他们不允许研究兴奋和抑制网络对 ictogenesis 的相互作用和协同作用。连续灌注脑切片与惊厥药物4AP 增强兴奋和抑制神经传递, 从而使研究急性 ictogenesis, 同时保持突触活动整体完整完好 12.
多边环境协定允许记录神经网络产生的电子活动从更多的观察点相比, 传统的细胞外场电位记录, 其中空间限制限制的电极数量, 可以在脑切片表面容纳。此外, 多边协定芯片提供了已知的电极间距离的额外优势, 这对于跟踪传播和评估记录信号的行进速度非常有用。虽然最初构思为记录从培养的神经元13,14, 多边环境协定现在也被用来表征从啮齿动物15和人类16获得的急性脑切片的电生理特征。因此, 在癫痫研究的背景下, 多边环境协定是一个有价值的工具, 用于查明神经网络在 ictogenesis16、17、18的核心中的相互作用。
然而, 在长时间 (几个小时) 实验协议中, 在获取或维持稳定的癫痫模式方面, 多边环境协定的记录本身就具有技术挑战。首先, 在大型和圆形浸没式记录室中, 组织氧合可能不足19,20典型的商用多边环境协定, 而低信噪比和温度不稳定可能影响记录的质量, 当一个高灌注率 (6-10 毫升/分钟) 是用来改善氧气供应的脑切片 (如请参见加热和灌注设备的技术说明21)。第二, 使用浸没式记录室19时很难观察到经常放电, 如癫痫放电等高频成分;当急性癫痫发作模式被化学诱导并涉及 ephaptic 机制时, 这种情况尤其如此, 因为4AP 模型22 (请参见11进行全面概述)。为了克服这些局限性, 研究人员提出了一些策略。例如, 增加灌注速率19、20 , 同时减少脑切片厚度 (≤300µM) 和区域17,18是实现组织充分氧气供应的关键因素。此外, 还可以通过使用穿孔的多边环境协定 (pMEAs) 来改善脑切片的生存能力, 从而使灌注的大脑组织从两侧18。
虽然所述方法大大提高了从脑切片记录的可行性, 但没有对长时间 (数小时) 记录和电刺激会话进行测试, 后者代表了一个重要大脑组织的压力。可能需要长时间的记录来研究癫痫模式的特定特征在时间上的演变, 这是不可能通过短期测量来揭露的。在星展研究的背景下, 可能需要长时间的实验协议来评估和比较几种刺激范式在同一脑切片中的作用。
当只有自发的电气活动需要记录时, 在数据分析过程中, 电极的映射通常是后验,即;相反, 对诱发反应或电调节范式的研究需要将刺激传递给特定的 ROI, 从而在实验过程中要求快速、容易的带电电极映射。
在这里, 我们说明了一个简单的实验协议, 允许诱导和维持一个稳定的 4 ap 诱发癫痫模式的成年啮齿动物脑切片。观察的活动忠实地再现了该模型的电生理特性, 其特点是使用常规的细胞外电生理技术。它持续了几个小时和经久持续的电刺激为重复的长的世纪。用于维持和孵化脑切片的分庭可以使用标准实验室用品 (图 1A) 轻松组装, 而一个允许最佳解决方案汇率和层流流的自定义记录室 (图 1B) 可以获得从商业来源或使用3D 印刷技术以实惠的价格。快速映射的 ROI (s) 的实时监测和选择的刺激电极是由一个自定义用户友好的 GUI 命名为mapMEA, 可以自由地根据请求提供。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57548/57548fig1.jpg"> 图 1: 用于此协议的自定义设备.(A) 在4AP 中, 用于恢复、预热和预孵化的保温室采用烧杯和培养皿组装而成。培养皿的直径应小于断路器, 并通过注射器柱塞来保持到位。培养皿的底部被一个尼龙网 (从柔软的丝袜) 所取代, 用氰粘在培养皿的边缘上。氧气通过弯曲的脊椎针 (22 克) 提供, 插入在培养皿和烧杯的壁之间。气泡应该从烧杯的侧面冒出来, 永远不会到达尼龙网, 以避免大脑切片移位。培养皿的顶部可以作为盖子, 以避免 ACSF 蒸发和保持氧气饱和度。(B) 自定义记录室。在: 入口水库容纳热化套管和参考电极。出: 出水口水库, 可容纳吸针。录音室。( C) 玻璃巴斯德吸管, 由火抛光卷曲, 用于处理组织并调整其在录音室中的位置。( D) 自定义切片保留锚点。(E) 安装在多边环境协定芯片上的记录室的最终组装。脑切片靠在固定位置的底部。红色箭头表示覆盖加热套管的聚四氟乙烯管, 而红色圆圈表示参考电极, 这是 ACSF 在进气库中浸没的饱和氯化钾颗粒。蓝色箭头表示出入口储层中的吸力针。<a href="/files/ftp_upload/57548/57548fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1112">
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		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Poly-D-Lysine&#160;</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">P7886</td>
			<td style="width:607px;">Needed for MEA coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td>S9888</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">P9541</td>
			<td>Chemical</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">795488</td>
			<td>Chemical</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2*2 H2O</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">C3306</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">RDD016</td>
			<td>Chemical</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">S5761</td>
			<td>Chemical</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2*6 H2O</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">M2670</td>
			<td>Chemical</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4*6 H2O</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">M5921</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">RDD023</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic Acid, 98%&#160;</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">107360</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-aminopyridine</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td style="width:127px;">A78403</td>
			<td>Convulsant drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STG-2004</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">STG4004-1.6mA</td>
			<td>4-channel stimulus generator with voltage (&#177;8 V) and current output (&#177;1.6 mA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">MEA1060</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">N/A</td>
			<td>MEA amplifier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:233px;">planar MEA</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">60MEA500/30iR-Ti w/o ring</td>
			<td>Must be without ring to allow using the custom recordingchamber</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:233px;">McRack</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;"></td>
			<td>Recording software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:233px;">McStimulus II</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;"></td>
			<td>STG4004 control software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">TC02</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">TC02</td>
			<td>2-channel thermostat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">PH01</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">PH01</td>
			<td>Heating perfusion canula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">MPH&#160;</td>
			<td style="width:145px;">Multichannel System</td>
			<td style="width:127px;">MPH&#160;</td>
			<td>Magnetic holder for PH01 and suction needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Elastostil E43</td>
			<td style="width:145px;">Wacker</td>
			<td style="width:127px;">E43</td>
			<td>Elastomeric sealant used to mount the custom recording chamber onto the MEA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">MEA Custom Chamber</td>
			<td style="width:145px;">Crisel Instrument</td>
			<td style="width:127px;">SKE-chamber MEA</td>
			<td>Custom recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;">Ag/AgCl electrode, pellet, 1.0 mm&#160;</td>
			<td style="width:145px;">Crisel Instrument</td>
			<td>64-1309</td>
			<td>Reference electrode for the custom recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Tergazyme</td>
			<td style="width:145px;">Sigma-Adrich</td>
			<td>Z273287-1EA</td>
			<td>Enzymatic cleaner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">MATLAB</td>
			<td style="width:145px;">The Mathworks</td>
			<td style="width:127px;"></td>
			<td>Programming environment for electrodemapping</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">VT1000S</td>
			<td style="width:145px;">Leica Biosystems</td>
			<td style="width:127px;">VT1000S</td>
			<td>Vibratome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;"></td>
			<td style="width:145px;">Warner Instruments</td>
			<td style="width:127px;">64-1309</td>
			<td>Ag-AgCl Electrode Pellet 1.0 mm (E205). Reference electrode.</td>
		</tr>
	</tbody>
</table>

recording and modulation of epileptiform activity in rodent brain slices coupled to microelectrode arrays

颞叶癫痫是最常见的部分复杂癫痫综合征, 对药物反应最少。深脑刺激 (DBS) 是一种有希望的方法, 当药理治疗失败或神经外科不建议。急性脑切片与微电极阵列 (多边环境协定) 相结合, 是研究神经网络相互作用及其电刺激调制的重要工具。与传统的细胞外记录技术相比, 它们提供了更多的观察点和已知的电极间距离的优势, 这使得研究的传播路径和电生理速度信号。然而, 在这一记录中, 组织氧合可能会受到很大的损害, 需要高的灌注率, 这是在实验温度降低信噪比和高振荡的代价。电刺激进一步强调脑组织, 使其难以追求长期记录/刺激时代。此外, 脑切片活动的电调制需要针对脑切片中的特定结构/通路, 要求电极映射在实验过程中能够轻松快速地进行。在这里, 我们说明如何使用平面多边环境协定对 4-氨基吡啶 (4 ap) 诱导的啮齿动物脑切片中的癫痫活动进行记录和电调制。我们表明, 从小鼠获得的脑组织优于大鼠脑组织, 因此更适合于实验。该协议保证了一种稳定的癫痫模式的产生和维持, 它能忠实地再现常规领域电位记录观察到的电生理特征, 持续数小时, 经久持续电刺激为长的世纪。由于使用小体积自定义记录室, 即使在低 (1 毫升/分钟) 灌注率下, 也可以实现整个实验的组织生存能力。实时监测和选择刺激电极的快速图象映射是由自定义图形用户界面 (GUI) 执行的。

Watch this Scientific Journal Video about Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays at JoVE.com

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

我们说明如何使用微电极阵列对 4-氨基吡啶诱导的啮齿动物脑切片中的癫痫活性进行记录和电调制。一个自定义记录室保持组织的可行性在长期的实验课。实时电极映射和激发对的选择由自定义图形用户界面执行。

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) ...

recording-modulation-epileptiform-activity-rodent-brain-slices

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.5K 次观看.  Istituto Italiano di Tecnologia. 我们说明如何使用微电极阵列对 4-氨基吡啶诱导的啮齿动物脑切片中的癫痫活性进行记录和电调制。一个自定义记录室保持组织的可行性在长期的实验课。实时电极映射和激发对的选择由自定义图形用户界面执行。

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

在普通马莫塞特中植入微电极阵列的立体外科（Callithrix jacchus）

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

摘要

探索更多视频

此视频中的章节

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

运行轮活动在啮齿类动物的昼夜节律的记录和分析

同时电生理记录和抑制剂在啮齿动物大脑微注射

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

海马鼠脑切片的突触密度评价

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

在普通马莫塞特中植入微电极阵列的立体外科（Callithrix jacchus）

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

摘要

探索更多视频

此视频中的章节

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

运行轮活动在啮齿类动物的昼夜节律的记录和分析

同时电生理记录和抑制剂在啮齿动物大脑微注射

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

海马鼠脑切片的突触密度评价

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

在普通马莫塞特中植入微电极阵列的立体外科（Callithrix jacchus）

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现