Name: cerebellar regional dissection for molecular analysis
Uploaded: 2020-12-05T14:30:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Cerebellar Regional Dissection for Molecular Analysis at JoVE.com

我们感谢Cvetanovic实验室的奥斯汀·费罗和朱奥-吉尔赫姆·罗莎在故障排除解剖、RNA提取和RTqPCR方面提供帮助。这项研究由M.Cvetanovic，R01 NS197387资助;HHS |国家卫生研究院。

1. 设置
<ol><li>收集必要的设备，包括斩首剪刀、钝钳、解剖剪刀、血管剪刀、微铲、下垂鼠脑基质、剃须刀刀片、200 μL 管尖、玻璃培养皿、玻璃滑梯和冰桶。将所有设备放在吸水垫上。</li>
	<li>将培养皿、玻璃板和大脑基质放在冰上。</li>
	<li>使用垂直角度的剃须刀刀片，修剪约 5 mm 的 200 μL 管道尖端。这使得尖端末端的开口大小大约为 1 mm 宽。这应该足以冲出 DCN 。但是，这也可以根据需要根据解剖进行调整。有许多提示，便于更换和调整。</li>
	<li>标记 1.5 mL 微融合管，具有动物识别和小脑区域（每只动物四根管）。</li>
	<li>提取后，用液氮填充冷冻安全容器以闪存冷冻组织。</li>
</ol>2. 大脑提取和解剖
所有实验都是根据明尼苏达大学动物护理委员会的指导方针进行的。
<ol><li>使用 5% CO2 暴露对鼠标实施安乐死。一旦呼吸停止，进行颈椎错位。用斩首剪刀斩首鼠标，并将尸体丢弃在适当的容器中。</li>
	<li>沿着头部的中垂线，从鼻子开始，一直往回走，用剃刀刀片切开。分离皮肤，分开到中线的两侧。使用剃须刀刀片切除两侧的肌肉，切开耳道。</li>
	<li>使用解剖剪刀，修剪任何脊髓区域，直至脑干与小脑相遇的地方，小心不要损坏小脑。</li>
	<li>将一个血管剪刀刀片插入脑干和椎柱之间的空间，切向耳道，用剪刀抬起来干净地切开骨头，但限制对组织的损伤。</li>
	<li>继续沿着头骨的边缘向嗅球切开，继续抬起来，同时切割以限制对脑组织的损伤。</li>
	<li>使用钝钳，轻轻剥去头骨的背面，露出大脑和小脑的后部区域。</li>
	<li>使用刚切开的头骨边缘的钝钳，将头骨的其余部分剥到大脑上。这一步应该去掉大部分的头骨帽，露出大脑。</li>
	<li>用血管剪刀和钝钳修剪头骨的其余部分，从大脑顶部清除大部分头骨。</li>
	<li>使用微铲，稍微抬起大脑，铲下并向上滑动，从剩余的头骨中取出嗅球，断开光路纤维。在这一点上， 大脑应该很容易自由。</li>
	<li>将大脑放入冰上，取出剩余的头骨或其他碎屑。</li>
	<li>使用微铲，轻轻地将大脑放入脑基质中，并向上倾斜。需要时间来确保它在矩阵中设置水平，特别是中线落在矩阵的中心。此基质专为成年小鼠脑组织设计，来自较年轻或患病动物的组织可能在基质中处于较低水平，但仍可在样本中实现可重复的结果。</li>
	<li>沿着下垂的中线放置一把剃须刀刀片，确保刀片一直推到矩阵底部（图 1B）。</li>
	<li>将另一把剃须刀刀片 1 mm 放在第一刀片的侧面（图 1B）。将另外两个刀片分开 1 毫米。最终结果应该是三个刀片放在大脑的一侧，所有1毫米分开。对另一边也做同样的事。总共将放置7个刀片（图1C）。</li>
	<li>小心地抓住剃须刀刀片的前端和后端，然后直接从矩阵中抬起来。剃须刀刀片外部的组织可以丢弃。</li>
	<li>慢慢地，一次将一把剃须刀刀片与其他刀片分开，小心不要损坏组织部分。</li>
	<li>小心地将组织部分从剃须刀刀片上滑落，然后用微型铲子滑到玻璃滑梯上。总共将有六个下垂的大脑部分（图1D）。</li>
	<li>4个最横向的部分将有DCN可见（紫色框， 图1D）。要隔离 DCN，将修剪过的 200 μL 管道尖端垂直固定在 DCN 上，并牢牢地向下推过组织，向四面八方摇晃，以完全解剖 DCN 与周围组织。直接抬起来，干净地去除DCN，并直观地确认组织在尖端的存在。</li>
	<li>将一根手指放在尖端顶部并向下推，导致组织凸出。将尖端放入正确标记的微融合管中，并确保将组织冲孔放置在管子底部。重复 2.17 用于其余三个部分，将 DCN 冲孔放在同一管中。将管子放入液氮中以闪烁冻结。 图1E中每拳大小的表示。</li>
	<li>提取了 DCN 的部分符合小脑半球的资格。推开这些部分小脑周围的脑组织其余部分。使用钝钳，轻轻地拿起这些半球小脑皮层部分，并放置到各自的微融合管。闪存冻结。</li>
	<li>对于最后两个真实部分（浅蓝色框， 图1D），推开周围的脑组织只留下小脑。使用剃须刀刀片，将前叶与后叶分离。切割应在球体 6 形成后，不应包括球状 10 （图1F）。</li>
	<li>使用钝钳，小心地将前小脑皮层部分和后小脑皮层部分放入各自的微充管中，将管子留在液氮中5分钟，以闪存冻结。从这里向前移动到RNA提取，或可能存储管在-80°C。</li>
</ol>3. RNA提取 
注:本协议是从冷泉港协议RNA提取与TRIzol20修改。TRIzol使生物材料溶解，从而有可能提取RNA。
<ol><li>将微融合管放入冰中，防止组织解冻过快，在微富管中涂抹 150 μL 的冷 TRIzol。与绝育害虫同质化。一旦组织被同质化，上下管道溶液，以确保没有剩余的组织完好无损。通过将小组织块拉入胰岛素注射器几次，进一步分解任何小组织块。</li>
	<li>再添加 350 μL 的 TRIzol 和管道上下混合彻底。让在房间温度下坐5分钟。</li>
	<li>在管子中加入 150 μL 氯仿，大力摇动，然后休息 2-3 分钟。氯仿将同质化组织溶液分相（RNA、DNA 和蛋白质）。</li>
	<li>离心机在 12，000 x g，在 15°C 下，10 分钟。确保所有管子都处于相同的方向。</li>
	<li>小心地取出管子，将离心机的温度设置为 4°C。仅将透明的交错阶段移入新管中（这是RNA），小心不要破坏不透明的相间（DNA）。 最低阶段为红色，含有蛋白质。管子中的剩余溶液可以保存或丢弃。</li>
	<li>以1:2的比例加入100%异丙醇（如果去除200 ul的水相，加入100微升异丙醇）。通过上下管道彻底混合。让在室温下休息10分钟。异丙醇使RNA沉淀出溶液。</li>
	<li>离心机在 12，000 x g，在 4°C 下，10 分钟。请务必将所有管子置于相同的方向，以便于可视化颗粒。产生的颗粒将是提取的RNA。</li>
	<li>小心地取出管子，用管道取出超强的管子，小心不要打乱颗粒。颗粒有点像凝胶状，很难看到，但应该能够根据离心机中管子的方向来估计它的位置。</li>
	<li>去除所有超高氨基剂后，在 4°C 下加入 500 微升 75% 乙醇、短暂涡流和离心机，7500 x g，4°C，5 分钟。乙醇进一步洗涤颗粒。</li>
	<li>小心地取出超高超，而不会破坏颗粒。让盖打开以干燥样品。这通常需要5-10分钟，但可以根据颗粒上剩余的乙醇量而有所不同。不要过干。</li>
	<li>干燥后，将颗粒重新注入无水区。在 DCN 样本中加入 20 μL，为所有其他样品添加 30 μL。</li>
	<li>重新暂停后，样品可以储存在 -80 °C 或进行进一步测试。</li>
</ol>4. 实时定量聚合酶链反应 （RTqPCR）
<ol><li>在此步骤之前，需要 DNase 治疗 RNA 以去除任何基因组 DNA，并利用 BioRad 中的 iScript 生成 cDNA。在制作 cDNA 之前，请务必使 RNA 的浓度正常化。此外，优化 qPCR 的引数也很重要。按照这里描述的方法完成这一步骤21。下面的 qPCR 的简要说明。</li>
	<li>引引器均从 IDT 购买。前向和反向序列都在同一个样本管中，并存储在 20 倍浓度下。 Rps18（控制基因）: 前进 - 5 '- 克塔加加特卡卡特 - 3' 反向 - 5 '- 阿卡卡塔克 - 3' 帕瓦尔布明（钙结合蛋白，抑制细胞）: 前锋 - '5 - 阿特加格特加加加格特克 - 3' 反向 - '5 - 阿格特克塔格 - 3' Kcng4 （钾通道子组）: 前进 - 5 '- CTTCTTCCTGTCGTGA-3' 反向 - 5 '- GCCCTCACTGTCAG-3' 阿尔多拉斯 C （泽布林二世， 小脑皮层的差别表达）: 前进 - 5 '- 阿加加加加塔塔特格 - 3' 反向 - 5 '- 塔格塔格特格特格克 - 3'</li>
	<li>qPCR 条件如下。预孵化期，5分钟，95°C。放大期，50 个周期:95°C 10 分钟，62°C 10 分钟，72°C 10 分钟。熔化曲线周期，5 秒在 95°C，1 分钟在 65°C，并将坡道速率设置为 0.07°C/秒，直到目标温度达到 97°C。然后冷却 10 分钟至 40°C。 所有 qPCR 反应均以三倍体进行，使用 2+ Ct分析基因表达，以 Rps18 作为负载控制，使基因表达正常化。散装小脑提取物用作参考。</li>
</ol>

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作者没有什么可透露的。

这里描述的方法使得评估四个不同的小脑区域内的基本基因表达和分子机制成为可能 - 深小脑核 （DCN）、前小脑皮层 （CCaV）、后脑脊柱皮层 （CCpV） 和半球小脑皮层 （CCH）。单独评估这些区域的能力将扩大我们对特定小脑区域异质性的了解，并可能揭示它们对各种行为的贡献。
通过下垂分割整个脑组织，可以很容易地可视化和识别小脑的这四个区域，从而允许快速解剖。?...

小脑包含超过一半的神经元在大脑中，历史上被称为运动控制和平衡中心在大脑1。最近，研究表明，小脑在认知、奖励处理等各种其他功能中起着关键作用，并影响2、3、4、5。
小脑有很好的解剖学:皮层区域由颗粒、普金耶和分子层组成。颗粒细胞形成颗粒细胞层，并通过平行纤维将输入发送到分子层的Purkinje细胞树突，该分子层的Purkinje细胞树突也从源自劣质橄榄的攀爬纤维中接收输入。Purkinje细胞向深小脑核（DCN）中的细胞发送抑制性投影，而深小脑核是小脑的主要输出。这种小脑回路的输出进一步调节在小脑皮层的抑制内周素的活动，包括高尔吉，石柱和篮子细胞4。这个小脑功能单元分布在小脑皮层的所有叶。尽管小脑的电路相对统一，但人类神经成像文献和患者研究表明小脑6、7的功能异质性。
小脑皮层可分为两个主要区域:中线定义的紫杉和横半球。紫杉可以进一步分为前叶和后叶。小脑的这些不同区域与促成不同行为有关。任务唤起或无任务的活动模式牵连到，前区域的vermis对运动功能的贡献更大，而后维米斯对认知6，7的贡献更大。真语也与影响和情绪有关，而小脑半球有助于执行，视觉空间，语言和其他助记符功能8。此外，解剖学研究提供了证据，功能上不同的小脑区域与不同的皮质区域9有关。病变症状映射显示，影响前叶（延伸至叶六）的中风患者在精细运动任务上表现较差，而后叶区域和半球受损的患者在没有小脑运动综合征10的情况下表现出认知缺陷。最后，区域小脑病理学表明，功能上不同的小脑区域也不同易患11、12病。
虽然探索较少，但初步证据表明，小脑皮质区域有不同的基因表达特征。Zebrin II的Purkinje细胞表达在动词中显示了区域特异性模式，因此后叶中有更多的泽布林II阳性细胞，前叶13中较少。这也与区域独特的生理功能相关，因为泽布林II负普金耶细胞显示更高的补品发射频率比Purkinje细胞是泽布林II阳性14。
除了小脑皮层外，小脑还包括作为小脑主要输出的深小脑核（DCN）。核由中间体 （MN）、插话 （IN） 和横向核 （LN） 组成。功能成像和患者研究表明，DCN也参与各种行为15，但很少有研究检查基因表达变化在DCN。
分子技术的进步使得评估大脑中的区域基因表达成为可能，并发现了生理学和疾病状态16中不同大脑区域的异质性。这种研究暗示小脑不同于其他大脑区域。例如，与其他大脑区域1相比，小脑中神经元与胶质细胞的比例是倒置的。即使在正常的生理条件下，与17个大脑区域相比，小脑中宣炎基因的表达也受到调节。分子技术在确定导致小脑疾病发病机制的途径方面也非常有用。例如，整个小脑提取物的RNA测序发现，与野生类型对照相比，在Purkinje细胞特异性转基因小鼠模型中，脊柱细胞1型（SCA1）中改变了基因。这些证据揭示了小脑Purkinje细胞中潜在的主要分子通路，并有助于确定潜在的治疗目标18。然而，最近的研究表明，小脑区域11、12、19的易患疾病的易患性存在差异。这可能表明，在不同的小脑区域发生了关键变化，这些变化可能被整个小脑提取物掩盖或未被发现。因此，有必要开发技术，使研究人员能够检查不同小脑区域的分子特征。
这里提出的技术描述了一种可重复的方法来解剖小鼠小脑的四个不同区域，以便将RNA与这些区域分离出来，并探索基因表达的区域差异。 图1A 中鼠标小脑的示意图突出了蓝色和黄色半球的真语。具体来说， 这种技术使得分离四个区域成为可能:深小脑核（DCN）（ 图1A中的红点盒），前紫杉（CCaV）的丘脑皮层（深蓝色） 图1A），后体的丘脑皮层（CCpV）（ 图1A中的浅蓝色）和半球的小脑皮层（图 1A中的黄色）。通过分别评估这些区域的基因表达，将有可能研究这些不同区域独立功能背后的分子机制，以及它们在疾病中的脆弱性的潜在差异。

<table><tbody><tr><td>1.5 Microcentrifuge tubes</td><td>ThermoScietific</td><td>3456</td><td></td></tr><tr><td>100% Isopropyl Alcohol</td><td>VWR Life sciences</td><td>1106C361</td><td></td></tr><tr><td>200 ul Pipet tips</td><td>GeneMate</td><td>P-1237-200</td><td></td></tr><tr><td>Adult Mouse Brain Matrix Sagittal</td><td>Kent Scientific Corporation</td><td>RBMA-200S</td><td></td></tr><tr><td>Blunt forceps</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>Macron</td><td>220905</td><td></td></tr><tr><td>Decapitation Scissors</td><td></td><td></td><td></td></tr><tr><td>Dissecting Scissors</td><td></td><td></td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Pharmco</td><td>111000200</td><td></td></tr><tr><td>Glass Slide (for electrophoresis)</td><td>BIORAD</td><td></td><td></td></tr><tr><td>Homogenizer</td><td>Kimble</td><td>6HAZ6</td><td></td></tr><tr><td>Ice Bucket</td><td></td><td></td><td></td></tr><tr><td>Insulin Syringe (.5ml)</td><td>BD</td><td>329461</td><td></td></tr><tr><td>iScript Adv cDNA kit for RT-qPCR</td><td>BIORAD</td><td>1725037</td><td></td></tr><tr><td>Micro Spatula</td><td></td><td></td><td></td></tr><tr><td>Needle Nose forceps</td><td></td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Pyrex</td><td></td><td></td></tr><tr><td>Primetime Primer for Aldolase C</td><td>IDT</td><td>Mm.PT.58&gt;43415246</td><td></td></tr><tr><td>Primetime Primer for Kcng4</td><td>IDT</td><td>Mm.PT.56a.9448518</td><td></td></tr><tr><td>Primetime Primer for Parvalbumin</td><td>IDT</td><td>Mm.PT.58.7596729</td><td></td></tr><tr><td>Primetime Primer Rps18&amp;nbsp;</td><td>IDT</td><td>Mm.PT.58.12109666</td><td></td></tr><tr><td>Single Edge Rzor Blades</td><td>Personna GEM</td><td></td><td></td></tr><tr><td>Sterile, sigle-use pestles</td><td>FisherScientific</td><td>12141364</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Ambion by Life technologies</td><td>15596018</td><td></td></tr><tr><td>Vascular Scissors</td><td></td><td></td><td></td></tr></tbody></table>

cerebellar regional dissection for molecular analysis

Cerebellum 在控制运动、平衡、认知、奖励和影响等几个关键功能中起着重要作用。成像研究表明，不同的小脑区域有助于这些不同的功能。研究区域小脑差异的分子研究是滞后的，因为它们大多是在整个小脑提取物上完成的，从而掩盖了特定小脑区域之间的任何区别。在这里，我们描述了一种可重复和快速剖析四种不同小脑区域的技术:深小脑核（DCN）、前体和后脑小脑皮层以及半球小脑皮层。剖析这些不同的区域可以探索分子机制，这些分子机制可能是它们对平衡、运动、影响和认知的独特贡献的基础。该技术还可用于探索不同小鼠疾病模型中这些特定区域的病理易感性差异。

在这些实验中，使用了四只11周大的雌性野生C57/Black6型小鼠。一只小鼠用于进行称为"散装小脑"的全小脑解剖，并允许将解剖区域的RNA水平与完全解剖进行比较。其他三只小鼠用于进行本协议中描述的小脑解剖。使用三只小鼠可以确保在小鼠身上检测到的RNA水平的趋势可重复。
图1A 代表小鼠小脑 - 蓝色和...

Watch this Scientific Journal Video about Cerebellar Regional Dissection for Molecular Analysis at JoVE.com

Cerebellar Regional Dissection for Molecular Analysis

不同的小脑区域被牵连到不同的行为输出中发挥作用，但潜在的分子机制仍然未知。本文描述了一种可重复和快速解剖半球、前部和后部区域以及深小脑核的方法，以便通过分离RNA和测试基因表达差异来探测分子差异。

分子分析的谷类区域解剖

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

cerebellar-regional-dissection-for-molecular-analysis

Research

JoVE Journal

Neuroscience

4.0K 次观看.  University of Minnesota. 不同的小脑区域被牵连到不同的行为输出中发挥作用，但潜在的分子机制仍然未知。本文描述了一种可重复和快速解剖半球、前部和后部区域以及深小脑核的方法，以便通过分离RNA和测试基因表达差异来探测分子差异。

视频: Cerebellar Regional Dissection for Molecular Analysis

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

科研

神经科学

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

将小鼠大脑显微切割成功能和解剖学上不同的区域

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

冷冻切片解剖成人室管膜下区，用于准确和深入的定量蛋白质组分析

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond its historical association with motor control. Structural and functional neuroimaging studies have further refined understanding of the functional neuroanatomy of the cerebellum beyond its anatomical divisions, highlighting the need for the examination of individual cerebellar subunits in healthy variability and neurological diseases. This paper presents a standardized pipeline for examining cerebellum grey matter morphometry that combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation (Automatic Cerebellum Anatomical Parcellation using U-Net Locally Constrained Optimization; ACAPULCO) and voxel-based registration of the cerebellum (Spatially Unbiased Infra-tentorial Template; SUIT) for volumetric quantification. 
The pipeline has broad applicability to a range of neurological diseases and is fully automated, with manual intervention only required for quality control of the outputs. The pipeline is freely available, with substantial accompanying documentation, and can be run on Mac, Windows, and Linux operating systems. The pipeline is applied in a cohort of individuals with Friedreich ataxia (FRDA), and representative results, as well as recommendations on group-level inferential statistical analyses, are provided. This pipeline could facilitate reliability and reproducibility across the field, ultimately providing a powerful methodological approach for characterizing and tracking cerebellar structural changes in neurological diseases.

A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification.

一种使用结构磁共振成像检查人小脑灰质形态测量的标准化管道

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

生物学

<meta charset="utf8"></head><body>小脑浦肯野细胞:器官型培养物中的 DNA 损伤反应

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

<meta charset="utf8"></head><body>作者聚焦:破译 ATM 在共济失调-毛细血管扩张症和相关小脑变性中的作用

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.