Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

这项工作得到了NIH、DA043982和DA046196的支持。

正如印第安纳大学机构动物护理和使用委员会（IACUC）和国家卫生研究院（NIH）指南所规定的，本研究中使用的动物以合乎道德和人道的方式对待。
注：所有工具和表面在开始任何工作之前，应用适当的溶剂清洗，以去除核酸酶18。
1. 储存大脑
<ol><li>使用常规方法13快速从安乐死成人CD1野生小鼠中取出大脑，体重约30?...

<ol>
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本研究描述了一种分离大脑小而特定区域，同时限制核酸和蛋白质降解的技术。一旦有机体死亡，对脑组织的损伤就会迅速发生。这是部分由于细胞外谷氨酸的快速积累和由此产生的兴奋毒性发生21。信使RNA特别容易受到降解22，23。22,蛋白质和核酸的分解在低温下大大减少，最近一篇文章描述了28，000年前冷冻在永冻...

本研究说明了用参考（如艾伦小鼠脑图集1）提取高质量核酸或蛋白质的冷冻大脑区域的剖面图。在这种技术中，大脑被闪速冷冻，储存在-80°C，以便以后在冷冻条件下进行解剖和解剖。这个过程允许研究人员在一个会话中收获大量的大脑，然后对其进行解剖，以便准确收集多个大脑区域。
在回答与基因和蛋白质表达有关的问题时，通常需要准确收集感兴趣的大脑区域（ROIs）。虽然药理学、电生理学和光遗传学可用于野生型或转基因啮齿动物，以帮助阐明支持观察到行为22、3、43,4的分子变化，但记录和蛋白酶诱导变化的测量常用于支持这些发现。定量逆转录聚合酶链反应（RT-qPCR）、西印、RNAseq 5、MAPSeq56和HPLC7等技术稳健，成本相对较低，使许多实验室能够研究小脑区2、4、5、64,5,6中的诱导分子2变化。
有几种方法可以,从大脑区域8、9、10、11、129,10提取和纯化核酸811或12蛋白质。许多实验室在收获99、1313时，通过冷却和在冰上切割大脑来收获大脑区域。虽然这种方法可以产生高质量的核酸和蛋白质，但它在一定程度上是有限度的，因为组织微环境中的降解可能发生在这些温度下。当尝试一次解剖大量动物或ROIs时，情况可能尤其如此。保持样品冷冻有助于保持实验室目标分子，同时为研究人员提供时间仔细比较每个部分两侧的地标，以收集相对纯净的样品。激光捕获是从大脑区域10收集RNA或蛋白质分析组织的另一种方法。此过程优于机械解剖，因为可以识别和隔离非常小且形状不规则的 ROI。然而，激光捕获受到昂贵设备和试剂的使用的限制，非常耗时，而且可能更容易样品降解。
冷冻组织的微冲压解剖并不新鲜。米克洛斯·帕尔科维茨等人的早期论文详细描述了基本技术14、15。,15本演示主要遵循原始工作，并进行了一些改进，以提高效率和降低所需设备的费用。例如，大脑部分是在冰冻的大脑块，而不是在低温。这将生成较厚的部分，从而减少收集 ROI 样本所需的部分数。此方法还解剖了位于绝缘盒内干冰上的冷冻玻璃板上的样品。这将在工作台的长凳上产生一个亚冻结阶段。以这种方式解剖的部分很容易操作，使研究人员能够将每个部分的两侧与参考进行比较，以限制来自所需 ROI 以外的区域的污染。
该协议的优点是：1） 大脑在整个过程中保持冷冻状态，这有助于保存蛋白质和核酸，并给研究人员时间仔细收获ROIs，2） 所需的试剂价格低廉，在大多数分子生物学实验室中都有发现。在这个过程中，整个大脑被分割到0.5~1.0毫米的大脑矩阵中，并放置在一个冷冻玻璃板上，不断用干冰冷却。在艾伦脑图集1或其他大脑地图集16，17,17中发现的地标用于识别感兴趣的区域，然后使用冷拳或手术刀进行解剖。由于组织从未解冻，以这种方式收获的区域为下游分析提供高质量的RNA和蛋白质。

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

随着我们对神经生物学的理解不断进步，分子分析通常对小大脑区域进行，如前额叶皮质（mPFC）或核积液。这项工作面临的挑战是解剖正确的区域，同时保留要检查的微观环境。在本文中，我们描述了一种使用大多数实验室中现成的资源的简单、低成本的方法。这种方法通过在整个过程中保持组织冻结来保存核酸和蛋白质。大脑使用大脑矩阵被切成0.5~1.0毫米的部分，并排列在冰冻的玻璃板上。每个部分中的地标都与参考进行比较，例如艾伦老鼠脑图集，并使用冷手术刀或活检冲孔对区域进行解剖。然后，组织在-80°C下储存，直到使用。通过这一过程，利用qRT-PCR和西式测定，成功地分析了大鼠和小鼠mPFC、核积液、背和内膜海马和其他区域。此方法仅限于大脑区域，可以通过清晰的地标识别。

为了验证这种方法，从成年CD1野生型雄性小鼠中前皮层采集，提取和特征蛋白质。RNA通过毛细血管电泳分析。降解RNA显示28S和18S核糖体带的强度损失，也显示降解产物作为25至200核苷酸之间的涂片（图5A，样本1）。优质RNA显示不同的核糖体带，在较低的分子量区域几乎没有或没有信号（图5A，样品2）。在此分析中也生成RNA完整性数 （RIN）。高于7的RIN表...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

此过程描述了离散冷冻大脑区域的收集，以便使用廉价和常用的工具获得高质量的蛋白质和RNA。

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 ...

此过程描述了离散冷冻大脑区域的收集，以便使用廉价且常用的工具获得高质量的蛋白质和RNA。由于大脑区域通过解剖保持收获冻结，目标分子被保留下来，研究人员有时间仔细解剖和储存感兴趣的区域。确定感兴趣的区域最初可能具有挑战性。使用常见的大脑地标，因为它们出现和退去的部分，相对于所需的区域将明确识别。从安乐死的成年CD-1野生小鼠身上取出大脑后，在液氮或异丙烷中闪冻结组织60秒。用干冰预冷，储存在零下80摄氏度。在解剖组织前24小时，将一个干净的大脑基质放在一叠解冻的冰柜包装上。将两个冰柜包之间的矩阵两侧夹到两者之间，确保在剃须刀槽底部和包装顶部之间留下大约半厘米。设置一个冷冻玻璃板进行解剖，将绝缘盒中冰块填充到距离顶部约五厘米处。然后在上面放置一层2.5厘米的干冰。用黑色塑料布盖住它。将玻璃板放在塑料上。和盘子边界周围的干冰。从冰柜中取出冷冻的大脑矩阵，将大脑皮层向上插入矩阵中。让组织与包装盒中的温度平衡 10 分钟。在此期间保持盖子打开。使用冷钳调整大脑在矩阵中的位置，使鼻窦和横向鼻窦与方块的垂直凹槽一起。一旦大脑就位，将一把冰冷的剃须刀刀片放在其中心附近，然后将其压入组织约一毫米。接下来，在大脑的每一端放置一个冰冷的刀片，然后一直向下按入矩阵。然后开始在旋转端添加冰镇刀片，一次放入一个插槽中，然后轻轻地将它们压入组织中一毫米。继续以一毫米间隔添加刀片，朝考达尔端工作。当所有刀片都到位时，用手指、手掌或钝器向下按压顶部，然后从一侧到侧面缓慢地摇动它们，以穿过组织。一旦刀片到达插槽的底部，抓住刀片组的每一侧，然后来回摇晃，将它们从矩阵中释放出来。释放刀片组后，将它们的旋转侧放在玻璃板上，将干冰放在堆栈旁边或顶部，以进一步冷冻样品，以便于分离。然后将具有锐利边缘的堆栈向下放置，并在拇指和手指之间移动堆栈来分离刀片。将玻璃板上的部分从旋转到骨面，通过将组织在手指之间弯曲或用第二个冰镇刀片分离，将组织从刀片中分离。有时，组织可能粘在刀片的两侧，必须注意保持到骨骼方向。在开始解剖之前，打开艾伦老鼠大脑图集或其他参考，并找到确定感兴趣区域所需的地标。使用冰镇钳或刀片翻转截面。并确保整个部分感兴趣的区域是一致的。用干净的手术刀或一拳切入部分，轻轻地将金属推入组织。来回摇动它， 使切割。收获感兴趣区域后，将其放入标有预冷却的 1.5 毫米管中，并将其存放在零下 80 摄氏度。要处理组织，加入冷 RIPA 缓冲液进行蛋白质提取或含有溶剂的瓜尼铀，并立即将其与玻璃羽化或机械均质器均匀化。为了验证这种方法，从成年CD-1野生雄性小鼠中采集了前额皮层，并进行了RNA和蛋白质的特征。当使用毛细管电泳分析时，降解的RNA显示28S和18S核糖体带的强度损失，以及25至200个核苷酸之间的涂片。虽然高质量的RNA在分子量较低的区域显示明显的核糖体带几乎没有信号。使用冷冻解剖方法获得的RNA与从新鲜收获的组织中准备的RNA进行比较。两种方法都产生高完整性和强核糖带的RNA。为了确认此方法可用于保存解剖组织的微环境以供以后分析，从已储存数周的解剖中前额皮质中提取了RNA。所有样品产生的高质量RNA具有明显的核糖体带和RNA完整性数超过8。为了确认冷冻解剖样品的蛋白质完整性，将蛋白质收集到硝基纤维素，并针对高分子量目标KCC2和较低分子量蛋白的检测。在所有情况下，频段都是尖锐和不同的，没有明显的分解产品。在整个过程中保持组织冻结非常重要，因为这样可以保留各部分的微环境，并保持部分形态，以便与大脑地图集图像进行比较。以这种方式收集的感兴趣区域可以在负 80 摄氏度下存储几个月，并可用于许多下游应用，包括 RT-qPCR、RNA-Seq、西印迹分析和 HPLC。这种方法已被用于探索接触THC和其他大麻素的围产期啮齿动物的肢体系统内的分子变化，以更好地了解这些药物如何影响大脑发育。

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

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

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...