Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

板20000分化的小鼠胚胎干细胞（MES）/孔在96孔聚-D-赖氨酸包被的板并在运动神经元的终末分化培养基维持5-7天。 1.复方管理和中毒与肉毒毒素/ A 执行下列所有工作在BSL2外壳，以保证符合CDC / NIH指南。 <ol><li>在聚丙烯96孔板中制备10mM储备溶液在100％DMSO中的每个库的化合物。使用10mM储备制备中间稀释板，其是10倍高于所需的筛选浓度。制备100μM的中间板通过分配10mM储备到96孔板中并用培养基稀释至100μM的。分配在细胞上11 10微升化合物对90微升的培养基。测试该化合物在10μM的最终浓度，并确保DMSO的终比例不超过0.5％。 </li><li>执行所有螺栓IES是利用活细胞和活性的毒素在生物安全二级条件。与使用12通道移液器手动80微升新鲜终末分化培养基代替旧的介质，在96孔板中。进行抽吸和分配步骤，按行，以避免细胞暴露而不媒体到环境空气中。 </li><li>预处理细胞之前通过加入10微升从源板使用12通道移液器，随后通过抽吸混合10倍的化合物应用毒素的化合物（两次）。对于每个96孔板中，治疗的6个孔的两列用10倍的DMSO稀释，在媒体上，作为高信号和低信号的控制。未经治疗的BoNT列显示全长的SNAP-25（高信号控制）的最高水平。对待其他列与肉毒毒素单独（无任何化合物）为低信号控制。使用低对照，观察SNAP-25和其检测与BACS的抗体的BoNT裂解的效率（ 图2）。 </li><li>孵育细胞30分钟，在37℃，6％CO 2，在细胞培养箱中的化合物。 </li><li>准备肉毒杆菌神经毒素A以上的磷酸盐浓度为1mg / ml的商业来源与终端分化培养基稀释缓冲液（PBS）到最终的10倍的股票工作。 </li><li>通过加入10微升10×的BoNT / A股成指定用于使用多通道移液管（ 图2）治疗和低信号控制井引发中毒。把指定为高的控制与单独媒体的井。 </li><li>净化所有的塑料制品和其他一次性用品，在水中至少20分钟的研究，浸泡在0.825％次氯酸钠溶液中谈到与肉毒毒素/ A的接触。请在生物安全柜的所有过程和​​两个前，用5％的洗涤剂消毒清洁剂实验如的MicroChem溶液后净化工作区域。 </li><li>标签板明确表示毒素的使用和incub吃板处理与1nM / L的BoNT / A 4小时，在37℃，6％CO 2下在细胞培养孵育箱。显示相应的指示牌，告知同事说，毒素是在实验室使用。 </li><li>停止BONT / A蛋白裂解甲醇固定。从各孔中除去含有毒素及化合物媒体用多道移液器，并丢弃在10％漂白溶液中。直接添加冰冷的甲醇（100微升）到每个孔中。 </li><li>培养板在室温（RT）下15分钟，以允许固定的发生。 </li><li>固定后，安全地处理废弃试剂（被认为中和）与生物安全1级协议和相应的丢弃。 </li><li>丢弃甲醇并用100微升PBS洗涤细胞并预先水化他们的免疫染色。执行两个额外的PBS洗涤。 </li><li>在最后一次洗涤后，留下的PBS中的孔中，密封板与微板粘合膜，并储存在4℃直至分析。商店板在4℃下FOř好几天。 </li></ol> 2.免疫染色免疫染色过程是劳动密集的，多步骤的操作，其包括重复的试剂分配/吸液循环和广泛板洗涤可导致潜在的引入显著板内和板间的变异性。一种半自动化的方法被应用到保存实验室人员的时间，提高测定吞吐量，并减少免疫染色可变性。 <ol><li>使用自动化工作站配备有96孔的头部能够传送量高达250微升并集成有机器人夹具臂移动至实验室器具插入模块甲板不同的位置（见材料和​​设备）来获得本协议。 <ol><li>模块化甲板被设计来承载不同类型的实验室器皿，并且可以在任一时刻支持多达11项。所述自动微处理程序配备了双杂志微孔板堆叠以保持和操纵高达每循环50的板。 </li><li>自动化工作站位于II级生物安全柜专为实验室自动化设备，以提供无菌和安全性的内部。用于自动免疫染色，在甲板上被安排为如图3所示，使试剂访问根据需要而无需人工干预。 </li></ol></li><li>预加载板根据需要为液体处理操作包含固定的细胞进入盘盒和传递到甲板上。使用96吸管头部装有250微升提示，从油井吸出PBS下降到10微升。透化细胞膜，促进抗体结合到与透化缓冲液（0.1％的Triton-X 100）的细胞内靶。之前以及将板返回到微孔板堆叠的第二存储盒为一温育15分钟，在室温（RT）下免除透化缓冲液（100微升）到每个。 </li><li>删除通透缓冲和PErform板用PBS洗涤（两次），为在步骤1.13如前所述。 </li><li>在最后一次洗涤步骤后，除去PBS缓冲液中，并分配100μl的含有在PBS中的1％马血清和0.1％吐温20封闭缓冲液中的成所有微孔板他们在RT返回到盘盒1小时温育之前。 </li><li>使用两个初级抗体为免疫染色:兔多克隆抗小鼠BACS的抗体，用于检测全长的SNAP-25和小鼠单克隆抗III微管蛋白抗体用于检测神经元（见材料和​​设备）的。温育60分钟后，除去封闭缓冲液，并分配50微升为4ug / ml的BACS的抗体和III微管蛋白的0.5微克/毫升的在封闭缓冲液入所有的板。 </li><li>孵育1小时在室温下除去第一抗体和洗涤用100μl的PBS中含有0.05％吐温（PBST）洗涤3次。 </li><li>使用抗小鼠IgG标记的Alexa Fluor 647以可视化的III微管蛋白和抗兔IgG标记的Alexa˚Fluor 568形象化BACS抗体。在封闭缓冲液中制备两种抗体为2微克/毫升的稀释和分配50μl的该混合物在每孔中。 注意:选择用于免疫染色的二级抗体标记有不同的荧光团，以允许检测其发射的荧光的使用高含量成像仪的检测器内的不同的信道不同的波长的光。 </li><li>保温1小时，在室温。吸出的第二抗体和洗涤3次，用100μl的PBST。 </li><li>最后一次洗涤后，加入100微升PBS含染料Hoechst公司（32微米）染色的DNA进行核检测。 </li><li>封板用不透光的薄膜，以保护从荧光漂白。板可被储存在4 &#160; °下星期没有显著信号丢失。 </li></ol> 3.影像注:使用高内涵成像Sys系统进行图像采集TEM（见材料和​​设备）。 <ol><li>规格高内涵成像定义的: <ol><li>选择板型，布局，字段数，目标:格雷纳ü明确，96孔板，16，分别为20倍的水。 </li><li>选择通道:核激发EX:405纳米通道4;细胞质EX:644纳米通道2;抗原特异性抗体频道EX:561纳米通道3，每个激光器的设置励磁电源，设置实验为2曝光，曝光1与一个激励，在644纳米，2曝光的两个激励在405纳米和561纳米。选择分级为斌。 </li><li>使用高对照孔用于与最大强度为SNAP-25信道和低对照孔为最小强度的曝光最优化。 </li><li>调整曝光，使每个信道的强度为约一半的饱和电平（2,000-2,500相对单位）。 </li><li>确定使用三角校正脚本小区蒙版通道的最佳Z平面。查看网络连接gure 5染色和成像后的结果。导出数据到其中的图像分析软件是驻留在服务器上。 </li></ol></li></ol> 4.图像分析（图6） 注:以下步骤描述了应用程序的哥伦布软件算法。 <ol><li>选择一个合适的算法来分割的主要对象（核）。如果需要的话，调整背景阈值和对比度参数。哥伦布软件提供4核的检测方法。选择的方法，该方法段准确细胞核通过目视检查分段核（F中igure 6a的方法M被选中）。 </li><li>选择突起算法（CSIRO轴突分析2）段中的突起如果需要，调整背景阈值和对比度参数来去除背景。参见图6b用于检测突起的参数。 </li><li>识别轴突区域（突起部分），BAS版上使用β-III微管蛋白的通道（Alexa的面粉640）作为掩模（ 图6c）的-3像素扩展的二次对象（突起）。 </li><li>计算信号通道（SNAP-25，（Alexa的面粉568）的突起区域和β-III微管蛋白通道（ 图6d＆6E）的强度。 </li><li>选择出口所需的参数，如神经突长度，SNAP-25的平均强度，β-III微管蛋白，核数，轴突段数等 （ 图1207米 ）。 </li><li>转让板使用机器人和负载到高内涵成像仪图像采集板堆垛。 </li></ol> 5.数据分析: 评估设计测定法的鲁棒性，计算从该板为基础的实验中使用以下参数。 <ol><li>信号（S）到背景（B）中:S / B =平均值高信号控制的强度）/平均值低信号控制的强度</li> &lt;利&gt;信号与背景的变化（CV）的系数（以测量特定信号的均匀性）:CV =（标准偏差（SD）的样品/平均值）×100（％），以确定在液体处理的可变性，试剂等</li><li>信噪比:S =（平均信号强度 - 指低信号控制的强度）低控制/ SD <ol><li>计算用的二分之一的96孔板的，而另一半的一个模拟中毒中毒的Z&#39;因子。 Z&#39;= 1 - 3 *（高信号控制+低信号控制的SD SD）/（平均高信号控制 - 低信号控制的意思）。用于筛选数据的进一步分析，正常化一些在平板上的基础的主原料的参数，以允许板之间和比较实验不同天之间。 </li></ol></li><li> ％的裂解，这反映了信号的全长度的SNAP-25的特异性抗体检测（BACS）相关联的降低:％裂解= 100％*（MEA高信号控制的n个强度 - 试样的强度）/平均高信号控制的力度。 </li><li>抑制裂解的百分比:抑制％= 100％*（样品的强度 - 指低信号控制的强度）/（平均高信号控制的强度 - 指低信号控制的强度）。 </li></ol>

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Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

肉毒杆菌神经毒素，相对缓解他们的武器化的高效力，导致其分类为A类（最高优先级）生物威胁剂由美国疾病控制和预防。不幸的是，没有FDA批准的治疗剂来对抗的BoNT中毒后的毒素已被内化的运动神经元。促进从肉毒毒素中毒的神经元恢复任何成药机制可能会导致对潜在疗法的发展对这种生物的威胁，以保护双方的军种和公众。在这篇文章中，我们提出了一个详细的分析HCI协议进行筛选的致命?...

细菌肉毒梭菌产生的肉毒杆菌神经毒素，对人类已知1是最有效的生物毒素之一。有7个不同的血清型肉毒毒素（肉毒毒素/ AG）。肉毒毒素/ A-胆引起麻痹的神经肌肉接头处由于SNARE复合体蛋白水解2,3。 SNARE蛋白水解阻止神经递质囊泡膜融合，从而阻止神经递质胞吐4。具体的SNARE目标取决于所涉及的中毒过程中的特殊肉毒毒素血清型。肉毒毒素/ A和肉毒毒素/ E切割SNAP-25，而肉毒毒素/ C切割两种SNAP-25和突触5。剩下的血清型切割突触（也称为囊泡相关膜蛋白（VAMP）。肉毒毒素/ A被选为试验发展，因为它是负责自然发生的肉毒杆菌中毒的比例很高，并具有行动6。发展小分子的最长期限治疗对肉毒毒素/ A是主要目标我们的药物研发项目，并利用传统的基于目标的方法来确定活性位点的蛋白水解酶抑制剂7，8-10。然而，创建活性位点抑制剂具有抗多种血清型和后曝光功效广谱活性的将可能是具有挑战性的。 因此，我们已经实现了一个创新的，表型的药物发现，使用肉毒毒素SNAP-25的切割作为一个功能的端点，以确定小分子，可以阻止肉毒毒素介导的运动神经中毒的方法。 SNAP-25所需的神经递质的释放，如SNAP-25的降解是预测性麻痹和致死的体内 。例如，基于细胞的筛选可能导致发现的负责毒素失活或抑制毒素途径的靶细胞内的细胞因子的新调节剂。在表型检测发展的一个重要因素是生理上相关的生物模型的选择。 W¯¯e和其他人描述了小鼠胚胎干（ES）细胞衍生的运动神经元概括初级运动神经元的免疫学性质，包括SNAP-25 -11- 13的表达。重要的是，这些蜂窝系统到的BoNT / A中毒高度敏感和演示的SNAP-25的剂量依赖性裂解响应于增加毒素11,12的浓度。在分化的运动神经元也产生在量，足以适用于高通量板为基础的分析，并允许细胞试验的阵列的设计。 表型分析是利用两种不同的抗体，肉毒毒素/鼠标运动神经元文化的陶醉在量化内源性表达的全长SNAP-25的裂解免疫荧光法。羧基末端肉毒毒素/ A裂解敏感（BACS）抗体只识别全长SNAP-25允许SNAP-25的肉毒毒素/ A介导的蛋白水解的评价在小鼠运动神经元表达10。的盐酸法的示意图示于图1。

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			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
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			<td style="width:119px;">3570</td>
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			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
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			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
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			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
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			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
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			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
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			<td height="21" style="height:21px;width:255px;">PlateStack</td>
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			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
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a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

从高和低控制数据创建两个不同的群体与两个位数超过3个标准差（图7A）的差。该筛选方法的目的是要找到样品群体中的化合物，其值越接近阳性对照群体，假定样品群体（ 图7B中，（i））内的正态分布。数据点，超出平均值的3个标准偏差被认为是从噪声统计上不同的，并且分类为有效的"命中"（ 图7B（ii）中 ，红色框）。被归类为"命中"化合物将受到在未?...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K 次观看.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

视频: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

科研

神经科学

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...