这项工作是由美国支持通过捷基金会，NARSAD，和NINDS授予R00NS064171 - 03。

原位脑固定1。 *准备一个10毫升注射器（28号针头）与磷酸充满缓冲液（PBS）。 *准备一个充满了4％多聚甲醛（PFA）的PBS 10 ml注射器（28号针头）。保留一个额外的PFA / PBS后固定5-10毫升。 <ol><li>实验小鼠腹腔注射致死剂量的Avertin或戊巴比妥。 </li><li>一旦鼠标镇静剂，湿用乙醇的腹部和安全鼠标与软木，蜡，或有机硅弹性体，使用夹层引脚分层的托盘底部。腹部朝上，安全的四个爪子表面 - 传播他们尽可能广泛。 </li><li>抓斗皮肤与胸骨水平钳，切十字花暴露肝脏。削减横向，然后通过肋骨和隔膜。拆下肋骨组织瓣，并继续切割，直到心访问。 </li><li>皮尔斯或剪断右心房漏传阅血液。 </li><li>通过左心室灌注的PBS冲洗循环系统残血。 </li><li>访问同一个针孔，整个鼠标的PBS / PFA随后通过左心室灌注修复。 </li></ol> 2。解剖和大脑提取<ol><li>皮肤切割耳道和眼轨道，向前拉动皮肤暴露头骨取出的头部，颈部，和头骨。 *步骤2.2）的说明，请参阅图2至2.5） </li><li>使用骨剪，切眼横向通过鼻甲骨骼的插座。每边。横切应当前的眼睛和嗅球。 </li><li>削减纵向从每个耳道眼圈。 </li><li>使用一个小解剖剪刀，切割枕骨板覆其他小脑从一个耳道。 </li><li>同一解剖剪刀，切割前方沿中线嗅球水平。 </li><li>使用镊子，从大脑中删除的每个骨板，照顾，以消除任何结缔组织，使大脑完好无损抬离骨时。 </li><li>剪断视神经和颅神经，切断上方的颈椎，然后取出固定液的大脑。 </li><li>修正后为1-2小时，在4 ° C洗3次，每次15分钟，用PBS。 </li></ol> 3。琼脂糖包埋<ol><li>熔体2％的高强度，低凝点琼脂糖类型IB（Sigma公司的产品目录号:A0576）在PBS生理盐水微波炉。 </li><li>熔化的琼脂糖转移到试管。试管放置在一个温暖的孵化器或水浴（40 ° -41 ° C），直至温度平衡。 </li><li>胶固定的脑组织上的氰基丙烯酸酯胶（ 图3A）的标本注射器的柱塞。刷饱和蔗糖PBS液对脑组织表面薄薄的一层，以方便以后的版本中的大脑切片的琼脂糖环。 </li><li>绘制成柱塞住房柱塞与脑组织（ 图3B）。 </li><li>移液器或倒入柱塞住房暖琼脂糖，占地脑组织。避免气泡捕获到琼脂糖。琼脂糖中的气泡会妥协切片质量。 </li><li>钳标本注射器冷（0 °至-25 ° C）寒蝉块1分钟，设置琼脂糖（ 图3C）。 </li></ol> 4。脑片切片的Compresstome <ol><li>组装Compresstome（ 图3D）将含有脑组织标本注射器。 </li><li>密切与试样的注射器插座（ 图3E）刀片的边缘对齐。 </li><li>用PBS液缓冲罐填充。 </li><li>千分尺一层厚旋转，拉伸试样的注射器琼脂糖脑组织块。 </li><li>按"开始"按钮控制单元的Compresstome启动切片过程。 </li><li>重复步骤4.4）4.5），直到获得所需的切片确定厚度。 </li><li>用刷子或吸管小瓶或填充染色，用PBS（ 图3F）井收获大脑切片。 </li></ol> 5。光学结算<ol><li>准备75％卷:卷的解决方案甘油:PBS。 </li><li>倒入（或吸管）关闭从收集到的大脑切片PBS冲洗量的一半。 </li><li>添加甘油/ PBS删除音量。 </li><li>允许温和的混合平衡在4 ° C，直到片下沉。 </li><li>重复步骤6.2）6.4），直到最后的甘油浓度达到75％。 </li><li> 90％甘油的PBS替代解决方案，并允许以平衡。 *请注意:小心不要引入气泡，而加入甘油的解决方案。 慢慢倒入解决方案，轻轻混匀，因为气泡会干扰统一equilibraTION和结转到幻灯片安装。 </li></ol> 6。切片安装成像<ol><li>切成一个开放的矩形双面密封胶（SA - S - 1L，格雷斯生物实验室），以适应一个微型幻灯片（Superfrost另外，25可视面积× 75 × 1毫米，＃48311-703，VWR猫。 ）与双方2-3毫米宽。 </li><li>脑片内的胶带，用吸管或画笔，矩形的中心广场。卸下温柔的共同愿望甘油过剩。 </li><li>幻灯片中所需的画笔秩序排列的大脑切片。 </li><li> ，轻轻地盖玻璃上的胶带（24 × 60毫米，猫。＃48383-139，VWR），注意不要引入气泡。轻轻地应用到玻璃盖的压力，以确保与胶带接触。 </li><li>吸从幻灯片和玻璃罩之间的边缘多余的甘油。 </li><li>密封幻灯片和盖玻片，用透明指甲油。 </li><li>晾干前成像，或长期存放在幻灯片框4 ° C。 </li></ol> 7。代表性的成果: 处理，成像，分析荧光标记的脑组织已经成为不可缺少的神经生物学研究。这些调查都需要复杂的遗传操作获得记者表达，在有针对性的神经元亚群，其次是低收入和高清晰度的图像分析。通常，实验和技术的限制使得不可能获得相同的动物的这些类型的数据，或在一个快速的方式。制备光清除，半厚的荧光成像艾滋病的大脑在这一挑战部分。例如设计一个完整的厚脑片标有狂犬病毒转基因EGFP的表达，使用啶和共聚焦显微镜和成像， 如图 4 所示，图5分别。对于啶成像，我们使用了徕卡M205 FA，和我们使用的Zeiss LSM 510共聚焦图像采集。由于相对简单的附加议定书，这种方法是能产生荧光记者表达了不到一天的周转时间组织有用的图像数据，并与低收入和高清晰度光学显微镜兼容。 如果研究者选择纳入额外死后标记方法，免疫组织化学染色，或较薄，协议相应延长。然而，上面介绍的方法代表了至关重要的记者表达完整的大脑简单和相对高通量筛选模式的方法。 <img src="/files/ftp_upload/2807/2807fig1.jpg" alt="图1"/> 图1。diagraming固定，解剖，切片，结算，半厚的脑片和安装程序的流程图。 <img src="/files/ftp_upload/2807/2807fig2.jpg" alt="图2"/> 图2。顺序切割的步骤中提取完整的小鼠大脑中的图。 <img src="/files/ftp_upload/2807/2807fig3.jpg" alt="图3"/> 图3的步骤安装和切片使用Compresstome的脑组织。一）脑组织的位置上切割柱塞使用强力胶。二）安装的脑组织进入柱塞琼脂糖嵌入画下来。三）巩固琼脂糖脑插件使用冷冻压缩块。四）琼脂糖大脑插件和柱塞插入到Compresstome切割室。 E）对齐的razorblade柱塞设备。 F）的大脑切片的切割和收集到Compresstome缓冲液槽。 <img src="/files/ftp_upload/2807/2807fig4.jpg" alt="图4"/> 图4:从甘油清除脑组织通过额叶皮质表达增强型绿色荧光蛋白（EGFP）的厚片的光镜图像。一）通过鼠脑冠状切片（厚200 UM）病毒载体表达EGFP标记，并在低的分辨率使用啶立体成像。比例尺，2毫米。 <img src="/files/ftp_upload/2807/2807fig5.jpg" alt="图5"/> 图5。荧光标记的第5层/ 6在清除厚的脑片皮层神经元的高倍率图像。一）高分辨率的共聚焦图像通过突出的地区（图4）的Z - Stack（厚150 UM）最大的投影。比例尺，25微米。

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	<li>Pfister, H., Lichtman, J., Reid, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Connectome Project. , (2009).</li><li>Briggman, K. L., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+neural+circuit+reconstruction+with+volume+electron+microscopy+techniques.">Towards neural circuit reconstruction with volume electron microscopy techniques.</a> Curr Opin Neurobiol. 16, 562-570 (2006).</li><li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Arenkiel, B. R., Ehlers, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+and+imaging+technologies+for+circuit+based+neuroanatomy.">Molecular genetic and imaging technologies for circuit based neuroanatomy.</a> Nature. 461, 900-907 (2009).</li><li>Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+the+genome+by+homologous+recombination.">Altering the genome by homologous recombination.</a> Science. 244, 1288-1292 (1989).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Shimomura, O., Johnson, F., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction,+purification,+and+properties+of+aequorin,+a+bioluminescent+protein+from+the+luminous+hydromedusan,+Aequorea.">Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.</a> J. Cell Comp Physiol. 59, 223-239 (1962).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+fluorescent+protein+as+a+marker+for+gene+expression.">Gene fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312, 217-224 (2006).</li><li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li></ol>

建强香港Precisionary仪器公司，即制造商在这篇文章中使用的产品的雇员。

鉴于广泛的应用，使用荧光蛋白为目标，通过光镜，需要迅速屏幕上，图像和分析完整的脑组织内神经网络已成为宝贵的调查的神经元亚群。 在用户友好的病毒载体，在体内电穿孔技术，转基因小鼠品系的发展技术进步提供了一个看似无限的源标记的细胞类型进行调查。然而，完整的脑组织图像分析仍是一个实验的瓶颈。这里介绍的方法已被证明为我们日常的脑组织?...

<table border="1"&gt;<tbody&gt;<tr&gt;<th&gt;该项目的名称</th&gt;<th&gt;公司</th&gt;<th&gt;目录编号</th&gt;<th&gt;评论（可选）</th&gt;</tr&gt;<tr&gt;<td&gt;骨剪刀</td&gt;<td&gt; FST</td&gt;<td&gt; 16044-10</td&gt;<td&gt;或同等</td&gt;</tr&gt;<tr&gt;<td&gt;清扫剪刀</td&gt;<td&gt; FST</td&gt;<td&gt; 14084-08</td&gt;<td&gt;或同等</td&gt;</tr&gt;<tr&gt;<td&gt; Ib型琼脂糖</td&gt;<td&gt;适马</td&gt;<td&gt; A0576</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Compresstome</td&gt;<td&gt; Precisionary仪器</td&gt;<td&gt; VF - 200</td&gt;<td&gt;其他vibratomes兼容</td&gt;</tr&gt;<tr&gt;<td&gt;双面胶</td&gt;<td&gt;格雷斯生物实验室</td&gt;<td&gt; SA - S - 1L</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Superfrost加幻灯片</td&gt;<td&gt; VWR</td&gt;<td&gt; 48311-703</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;玻璃盖</td&gt;<td&gt; VWR</td&gt;<td&gt; 48383-139</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;甘油</td&gt;<td&gt; EMD的化学品公司</td&gt;<td&gt; GX0185 - 6</td&gt;<td&gt;或同等</td&gt;</tr&gt;</tbody&gt;</table&gt;

a rapid approach to high-resolution fluorescence imaging in semi-thick brain slices

基础和临床神经科学的根本目标是为了更好地理解人的身份，分子结构和特点在正常和病变脑的神经元的连接模式。朝着这个，一个很大的努力一直放在建设高解析度神经解剖学地图1-3。随着分子遗传学和光镜的进步已经扩展能力查询，不仅神经元的形态，而且分子和细胞构成的单个神经元及其相关的网络4。标记和操纵通过转基因和基因打靶技术在啮齿类动物的神经元的能力的重大进展， 现在允许以5-6将"纲要"神经元亚群的调查。可以说，最有影响力的现代神经科学的贡献之一，已发现和克隆基因编码荧光蛋白（FPS）7-8，沿着他们的后续工程，在海洋无脊椎动物的产量不断扩大的一个至关重要的记者工具箱9。现在利用特定细胞类型的启动子活性的驱动器在离散的神经元群体针对性的FP表达给予神经解剖与遗传精密的调查。 工程FP神经元中表达，大大提高了我们的大脑结构和功能的认识。然而，成像的单个神经元和脑深部组织，或在三个方面及其相关的网络，仍然是一个挑战。由于脂肪含量高，神经组织，而不透明和展品自动荧光。这些固有的生物物理特性，很难想象和高分辨率的图像使用超过几十微米的深度标准啶或共聚焦显微镜荧光标记的神经元。为了规避这一挑战调查往往采用串行薄片成像和重建方法10，或双光子激光扫描显微镜11。目前这些方法的缺点是相关的劳动密集的组织准备，或成本高昂的仪器。 在这里，我们提出了一个相对快速和简单的的方法来可视化在固定半厚的小鼠大脑切片荧光标记的细胞，通过光学结算和成像。在附加协议中，我们描述的方法:1）固定在原地通过intracardial灌注，2）剥离和清除整个大脑，3）固定大脑包埋在琼脂糖，4）精密半厚片准备使用新vibratome仪器的脑组织光镜和Z - Stack的重建（ 图1），5）通过甘油梯度结算脑组织，以及6）安装在载玻片上。 准备脑切片中，我们实现了一个相对较新的仪器，称为"Compresstome"，VF - 200（http://www.precisionary.com/products_vf200.html）。这台仪器是一种半自动切片机，配备一个电动推进和叶片振动系统与功能，在功能类似于其他vibratomes。与其他vibratomes不同的是，要切片的组织琼脂糖插件是安装在一个不锈钢圆筒内。该组织是在想要从汽缸厚度挤压，并向前推进的振动刀片切割。琼脂糖插件/气缸系统可重复性组织安装，对齐方式和精密切割。在我们手中，"Compresstome"产量高品质的电生理，免疫组织化学组织切片，并直接固定组织安装和成像。与光学清算相结合，在这里我们展示高分辨率荧光成像的固定半厚的脑片的准备​​。

Watch this Scientific Journal Video about A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices at JoVE.com

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

在这里，我们描述了一个快速和简单的的方法，形象荧光标记的细胞在半厚的脑片。通过固定，切片，和光清除脑组织中，我们介绍了如何标准啶或共焦成像可用于个人完整的神经组织内的细胞和神经网络的可视化。

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

a-rapid-approach-to-high-resolution-fluorescence-imaging-semi-thick

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

16.2K 次观看.  Baylor College of Medicine (BCM). 在这里，我们描述了一个快速和简单的的方法，形象荧光标记的细胞在半厚的脑片。通过固定，切片，和光清除脑组织中，我们介绍了如何标准啶或共焦成像可用于个人完整的神经组织内的细胞和神经网络的可视化。

视频: A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

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 ...

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 ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

一个快速高分辨率的荧光成像的方法在半厚的脑片

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