Name: generation and on-demand initiation of acute ictal activity in rodent and human tissue
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Description: Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

这项工作得到了加拿大卫生研究所 (mop 119003 至 peter l. carlen 和 taufik a. valiante)、安大略省大脑研究所 (致 taufik a. valiante) 和 mititex 学生研究补助金 (给 michael chang) 的支持。我们要感谢利亚姆·龙在拍摄视频手稿方面的协助。我们要感谢 paria Baharikhoob、abeeshan selvabaskaran 和 shadini dematagoda 协助汇编这份手稿中的数字和表格。图 1a、 3 a、 4 a和6 a都是根据 chang 等人公布的数据得出的原始数字.16岁

所有涉及病人的研究都是根据大学卫生网络研究伦理委员会根据《赫尔辛基宣言》批准的一项议定书进行的。涉及动物的程序符合加拿大动物护理理事会的准则, 并得到克雷姆比尔研究所动物护理委员会的批准。
1. 第一议定书: 急性体外缉获模型
<ol><li>解剖溶液与人工脑脊液的制备
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<li>使用气石气泡扩散器 (水轮空气炉), 用碳原 (95% o2/5% co2) 过滤碳化水...

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脑片用亲惊厥药物或改变的 acsf 灌注剂治疗, 以增加神经网络的兴奋性, 促进冰门事件的沉淀 (电图癫痫发作事件)。对于小鼠, 体质运动区的首选冠状切片应包含扣带回皮层, 区域 2 (cg), 但不包含视网膜平底区 (rs);这些解剖标记有助于确定最适合诱发冰毒事件的冠状切片的范围。小鼠组织的一个可选的修改是将大脑切片的两个半球切成两半, 用于匹配对实验设计, 因为这两个半球几乎是相同的 (类似?...

急性癫痫发作模型可以成功地再现断层特征, 使人想起在癫痫发作者的脑电图 (eeg) 中观察到的冰毒事件。研究人员使用这些类似于冰的事件 (这里称为 "冰形事件") 作为癫痫发作事件1的代孕者。临床上, 冰体事件是癫痫发作事件的可靠代名词, 因为癫痫发作是一种源于大脑的神经紊乱。在癫痫监测单位, 神经学家依靠对伊卡事件的检测来确认大脑的癫痫发生区, 并将其分离出, 使其切除2。在重症监护室, 医生监测精神病活动, 以评估是否有任何癫痫发作的病人持续的癫痫发作活动3。控制癫痫发作仍然是医学界面临的一个挑战问题, 因为30% 的癫痫患者对现有药物4、5和10% 的涉及药物引起的癫痫发作的病例具有耐药性。对标准治疗没有反应3。这引起了社会的严重关切, 因为10% 的美国人在一生中被预测会经历一次癫痫发作事件, 3% 的人预计会患上癫痫.
研究慢性癫痫模型中的癫痫发作是昂贵的, 费力的, 往往需要几个月的时间来准备7。在自由移动的动物中进行电生理记录也是很困难的。人体临床试验面临着类似的问题, 以及与患者同意、参与者背景的差异以及涉及道德和伦理方面的考虑等额外的复杂性。另一方面, 急性癫痫发作模型是有利的, 因为它们的制备相对方便, 成本效益高, 能够产生大量的冰毒事件用于研究9。此外, 组织被固定在一个稳定的位置, 所以条件是理想的执行必要的电生理记录, 研究癫痫发作动力学和相关的潜在病理生理学。急性癫痫发作模型在硅胶(计算机) 模型中仍然是有利的, 因为它们是基于由大脑组成的神经网络组成的生物材料及其所有固有的因素和突触连接, 这些物质可能无法捕获即使是最详细的计算机型号10。这些特点使急性癫痫发作模型能够有效地筛选潜在的抗癫痫疗法, 并在推进这些疗法以进一步调查慢性癫痫模型和临床试验之前作出初步调查。
通常情况下, 急性癫痫发作模型来自正常的脑组织, 已遭受高度兴奋的条件。要诱导健康脑组织中的临床相关的冰体事件, 重要的是要了解大脑在静态11中的最佳功能, 在这种状态下, 兴奋 (e) 和抑制 (i) 是平衡的 12。e-i 平衡的中断可能会导致高度可激发的发作状态, 在这种状态下, 冰体事件会沉淀。因此, 在这一概念框架内, 有两种主要策略可以在大脑切片 (体外) 或全脑 (体内) 制剂中产生冰毒事件: 要么减少抑制 ("抑制"), 要么增加抑制。激励 ("非抑制")。然而, 冰印象事件是高度有序和同步的事件, 需要 gaba中西神经元的影响来协调神经网络活动13,14。因此, 非抑制模型是在孤立的神经网络中产生冰毒事件的最有效的模型, 例如在体外大脑切片15中, 而体外抑制模型通常会导致尖峰活动让人想起像兴趣的尖峰此外, 在此概念框架内, 瞬时同步事件还可以可靠地触发 ictal 事件16。事实上, 当在临界状态转换 ("分叉") 第18点时, 应用于神经系统 17的任何微小扰动都可能触发冰形事件.传统上, 这些扰动是由电刺激引起的。然而, 最近神经科学中光遗传学的发展, 现在为诱导临界状态转变提供了更优雅的策略.
本文所述的方法演示了如何在体外 ( 《议定书》第1步) 和体内研究 (《议定书》第2步) 的急性发作模型中按需生成冰毒事件。它们涉及大脑区域的选择、癫痫诱导方法、研究类型和物种;然而, 重点将是推荐的选择急性4-ap 皮质癫痫发作模型, 因为它的多功能性在广泛的研究类型。急性体外 4-ap癫痫发作模型是基于标准的方案, 为电生理记录和成像研究准备高质量的脑片19。这些方案已经被用来从 16,20和人类21的体生长运动皮层的体外冠状大脑切片。在这些类型的大脑切片中产生冰毒事件的修改以前已经演示过16 , 下面的《议定书》描述了全部细节。急性体内4-ap 皮质发作模型是基于标准的协议, 准备开颅术的影像学研究22。修改是, 开颅后没有安装 (玻璃滑梯) 窗口。相反, 前惊厥药物 (4-ap) 局部应用于暴露的皮层, 以诱导在动物全身麻醉期间的冰毒事件。据我们所知, 我们的小组是第一个在1 6、23 小鼠体内开发这种急性皮质癫痫发作模型的人。建立了以成年小鼠为研究对象的体内急性4-ap 皮质发作模型, 以补充幼龄组织的体外切片模型。在成人体内癫痫发作模型中复制发现有助于通过解决2d 脑切片 (与三维全脑) 非生理条件的内在担忧来概括切片模型的发现结构) 和幼体与成人组织之间的生理差异。
按需的气门事件启动方法被证明使用的神经递质与皮科斯普利或光遗传学策略。据我们所知, 我们的小组是第一个通过皮科普利策16使用神经递质在人体组织中引发冰毒事件的组织。在光遗传策略中, c57bl6 小鼠菌株是用于表达转基因的常规菌株。通道罗多普辛-2 (chr2) 在 gabaep 能间质间质细胞或谷氨酰胺锥体细胞中的表达将提供可选的能力, 在短暂的光脉冲下按需生成冰门事件。合适的光遗传学小鼠菌株包括商业上可获得的 c57bl6 变种, 该变种使用小鼠泡状 gaba 转运体启动子 (vgat) 24 或锥体细胞, 使用小鼠胸腺细胞抗原 1,在任何一个神经元中表达 chr2促销员 (thy1)25。这些市售的 vgate-chr2 和 thy1-chr2 小鼠提供了机会, 分别激活 gabaep 神经元或谷氨酸神经元, 在新皮层与蓝色 (470 纳米) 光。在急性癫痫发作模型中, 按需生成冰毒事件的能力可以为研究癫痫发作的启动动力学和有效评估潜在的抗癫痫疗法提供新的机会。

<table>
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		<tr>
			<td>Sodium pentobarbital</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>1 mm syringe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>25G 5/8&#8221; sterile needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Single edge razor blade (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Instant adhesive glue</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Glass petri dish (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Splinter forceps (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>PVC handle micro spatula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Micro spoon with flat end</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Detailing brush 5/0</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purcahsed from a boutique art store</td>
		</tr>
		<tr>
			<td>Wide bore transfer pipette</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dental Tweezer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Thermometer (digital)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased on Amazon.ca</td>
		</tr>
		<tr>
			<td>Check carbogen tank (95%O2/5%CO2)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>brain slice incubation chamber (a.k.a. brain slice keeper)&#160;</td>
			<td>Scientific Systems Design Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4 H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (HNaPO4&#183;H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2&#183;2H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
	</tbody>
</table>

generation and on-demand initiation of acute ictal activity in rodent and human tissue

控制癫痫发作仍然是医学界面临的一个具有挑战性的问题。为了取得进展, 研究人员需要一种方法来广泛研究癫痫发作的动态和研究其潜在的机制。急性癫痫发作模型很方便, 提供了进行电生理记录的能力, 并能产生大量的电测事件。然后, 急性癫痫发作模型的有希望的发现可以推广到慢性癫痫模型和临床试验。因此, 研究急性模型中的癫痫发作, 忠实地复制临床癫痫发作的电特征和动态特征, 对于得出临床相关的发现至关重要。研究从人体组织制备的急性癫痫发作模型中的冰毒事件对于得出临床相关的结果也很重要。本文的重点是皮质4-ap 模型, 因为它在体内和体外研究中, 以及在小鼠和人体组织中产生冰体事件的多功能性。本文中的方法还将描述使用零 mg2 +模型进行癫痫发作诱导的另一种方法, 并详细概述不同急性中产生的癫痫样活动的优点和局限性。扣押模型。此外, 通过利用市售的光遗传小鼠菌株, 可以使用短暂 (30 毫秒) 的光脉冲来触发与自发发生的相同的冰体事件。同样, 30-100 毫秒的神经递质 (伽玛-氨基酸或谷氨酸) 可应用于人体组织, 以引发与自发发生的事件相同的冰门事件。在急性癫痫发作模型中按需触发冰门事件的能力提供了新发现的能力, 可以观察作为癫痫发作起始动力学基础的事件的确切序列, 并有效地评估潜在的抗癫痫疗法。

将 100μm 4-ap 应用于来自幼龄 vgat-chr2 小鼠的高质量 (无损坏) 的大脑片, 可在15分钟内可靠地诱导复发性的冰门事件 (> 5秒) (图 1 ai)。将 100μm 4-ap 应用于劣质切片, 导致了爆裂事件或尖峰活动 (图 1Aii)。平均而言, 每个解剖的老鼠大脑中的40% 的切片成功地产生了冰毒事件。此外, 83% (25/) 的解剖小鼠导致至少一个大脑切片成功地产生?...

Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

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

急性癫痫发作模型对研究癫痫发作事件的机制很重要。此外, 按需生成癫痫形成事件的能力提供了一种高效的方法来研究其启动背后的事件的确切顺序。在这里, 我们描述了在小鼠和人体组织中建立的急性 4-氨基吡啶皮质发作模型。

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 ...

这种方法可以帮助回答癫痫发作机制研究中的关键问题，例如，什么神经亚细胞负责电图发作发作和终止。这种技术的主要优点是，它可以复制电学发作事件，按需，在体内和体外模型，让人想起那些观察到的临床。这种技术还可用于寻找潜在的抗癫痫疗法，通过尝试触发电图癫痫发作事件，在不同的抗癫痫药物候选者的应用。要准备皮质切片，请使用即时粘合胶将临床前脑组织粘在振动器阶段。轻轻地将规格支架放入缓冲盘中。确保大脑的后部部分面对振动器的刀片。将大脑切成450微米厚的部分，在后腹方向。在临床前动物大脑中进行第一次切口，以去除嗅觉球。然后，进行后续切割，直到观察到躯体感官运动区域。使用宽孔移液器。将目标日冕切片转移到含有冷解剖溶液的培养皿中。对于日冕切片，在新皮质小切口下方进行横向切割，然后在中线切割以分离半球。然后，使用新的剃刀刀片从切片中切割任何多余的组织。有效执行大脑切片程序至关重要。每一刻脑组织不淹没在ACSF中，都有害于其质量。受损、质量差的脑组织不太可能产生电学发作事件。将含有新皮层的日冕片的后侧部分转移到第二个装满35摄氏度ACSF的Petrie菜中，一会儿。然后，立即将切片转移到含有35摄氏度碳化ACSF的孵化室。将大脑切片在35摄氏度下稍微浸入孵化室30分钟。然后，从水浴中取出孵化室，让其回到室温。等待一个小时，让大脑切片恢复，然后再进行电生理记录。在此过程中，切出略大于脑片的镜头纸。使用宽孔移液器或细节刷将大脑切片转移到使用牙科钳子放置的切出镜头纸上。然后，将带脑片的镜头纸传输到录音室，然后用竖琴屏幕将其固定到位。随后，以每分钟3毫升的速度，在记录室中用碳化ACSF将大脑切片以35摄氏度的速度大量使用。使用数字温度计确保记录室温度为 33 至 36 摄氏度。然后，使用汉密尔顿注射器将玻璃电极回填为 10 微升的 ACSF。在20次立体显微镜下，使用手动操纵器将记录玻璃电极引导进入表面皮质层，二、三。使用标准软件查看大脑切片的电活动。为了诱导电图癫痫发作样的活动，在100微摩尔时用ACSF含有4-AP的脑片。使用标准软件查看大脑切片的电活动。要生成电图癫痫发作样事件，使用光遗传学小鼠大脑切片的光遗传学策略，使用手动操纵器将直径为 1，000 微米的光纤直接位于记录区域上方。应用蓝光的短暂脉冲以启动 ictal 事件。专门开发了一个用户友好的 MATLAB 程序，用于检测和分类体外和体内 4-AP 癫痫发作模型中发生的各种类型的表皮事件。此检测程序可从 Valiante 实验室 GitHub 存储库获得。将100微摩尔4-AP应用到优质的450微米大小的皮质脑切片中，从幼年VGAT通道霍多普辛小鼠，在15分钟内可靠地诱发复发性皮质事件。将100微摩尔4-AP应用到质量差的切片上，导致爆裂事件或尖峰活动。平均而言，每个解剖的临床前大脑的切片有40%，成功地生成了像ictal这样的事件。此外，83%的解剖小鼠至少产生一个脑片，成功生成了像ictal这样的事件。在具有自发发生的类似ictal事件的大脑切片中，在大脑切片上应用一个30毫秒的光脉冲，可靠地触发了在形态学上是相同的类似ictal事件。同样的发现是在Yy One Channelrhopsin两只老鼠的大脑切片中。因此，无论激活哪个神经元子组，隔离的皮质神经网络中的任何短暂同步事件都导致一个与ictal一样的事件出现。这些像ictal这样的事件包括哨兵尖峰、补品式射击、克隆式射击和接近尾声的爆发式活动。它们的性质与与临床癫痫发作相关的电图特征相似。按照这个程序，其他类型的大脑状态转换可以进行，以解决问题，如，什么是大脑状态转换背后的神经生物机制？我们如何调节这些转变，以防止进入各种病理脑状态？

Research

JoVE Journal

Neuroscience

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

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

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

测量小鼠使用车轮运行活动的昼夜和急性对光反应

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

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

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

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

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

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, ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

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

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) ...