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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 Microcentrifuge tubes</td>
			<td >ThermoScietific</td>
			<td >3456</td>
			<td ></td>
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		<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&#62;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&#160;</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>
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		<tr >
			<td >TRIzol Reagent</td>
			<td >Ambion by Life technologies</td>
			<td >15596018</td>
			<td></td>
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		<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

3.8K 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

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

一个简单的综合评分系统评估小脑共济失调的小鼠模型的表型

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

科研

神经科学

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...