佩德罗巴里DE LA的Maza基金会支持这项工作。

1）鱼藤酮灌胃<ol><li>称量动物。 </li><li>计算总量的鱼藤酮/车辆所需的解决方案。在这种情况下，每克动物体重0.01毫升。 </li><li>与以前计算的鱼藤酮溶液体积（0.625毫克/毫升鱼藤酮，4％的羧甲基纤维素钠和1.25％，氯仿）或车辆的解决方案（4％羧甲基纤维素钠和1.25％，氯仿），充电1 mL注射器。在这种情况下，对应于5毫克/公斤的鱼藤酮剂量。 </li><li>拿起鼠标，一只手​​握住鼠标的尾部，舒展，另一方面与前两个手指拉颈部的皮肤。 </li><li>一旦头部是固定的两根手指之间，靠在手掌，并把它上下颠倒。修正使用第四个和第五个手指的尾巴。 </li><li>保持对后面的头，按上腭，打开鼠标的嘴。轻轻地引入口灌胃。重要的是要使用一个灌胃针，而不是一个普通的针。慢慢地，在胃远远不够的方向移动灌胃。这将避免材料在肺部获得。 </li><li>鱼藤酮溶液或车辆管理轻轻按下栓子。完成后，慢慢地提取物灌胃。 </li><li>从一个笼子里的所有动物都将在另一个笼子里的动物，直到完成。 </li></ol> 2）免疫染色程序<ol><li>治疗后，小鼠的麻醉使用氯胺酮100毫克/公斤和Xylazin 10毫克/公斤的IP和intracardially灌注4％多聚甲醛的PBS。 </li><li>的胆量，然后由夹层中提取，放置过夜，并在15％的蔗糖。第二天，组织转移30％的蔗糖和4℃过夜孵育° C。 </li><li>组织，然后放置在组织- TEK，并储存在-80℃冷冻，直到使用。 </li><li>使用低温恒温器，20微米肠道截面转移到Superfrost幻灯片。 </li><li>阻塞和使用反βIII-微管蛋白和抗-α-突触核蛋白抗体的免疫程序，然后执行 9如前所述。 </li></ol> 3）α-突触核蛋白共享图像分析建议外部图像“出身的人不知道执行的分析。因此，该数据应该加以编纂。使用斐济软件进行图像分析。 <ol><li>打开的共聚焦图像文件。然后，分裂的渠道，因此，它是可以对每个通道的独立工作。 </li><li>关闭这些渠道，不会使用和选择分析&gt;设置规模和像素的大小设置为1。已知的距离设置为0.13（这将取决于客观和使用的决议），点击全球。现在将显示图像的大小在微米。 </li><li>选择α- synuclein的通道，并选择“节”，确保选择在所有切片的神经节封闭。 </li><li>按“图像”&gt;“重复重复的图像。随着节选定的，只是一个形象的神经节的大小将被复制。 </li><li>按“编辑”&gt;“清除外。选定区域之外的形象的一部分将变成黑色。 </li><li>再次重复的形象，在后面的步骤，程序，以确定。图像具有较高的信号噪声背景的比例不应该用于图像分析。 </li><li>选择图像&gt;调整&gt;阈值，然后选择MaxEntropy。然后移到裁剪图像和原片，以确保过程顺利。熵阈值选择一个基于图像的直方图，只有那些与强度高于此阈值将在白色比黑色图像显示的像素的亮度信号电平。 </li><li>选择过程，然后在平滑。这一步变换的图像边缘平滑pixeled的边缘。 </li><li>选择过程，然后对二进制，并选择二进制。图像将显示为黑色比白色图像。 </li><li>重复的图像复制的图像上执行下一步。 </li><li>点击颗粒分析&gt;分析。然后选择“显示：”“纲要”，并点击总结。其余的设置应在默认阔叶。此功能承认一个信号，这些组合在一起的像素总数。总表面的信号加像素的群体数量和平均粒径的像素。 </li><li>比较和选择最大的片，调整好。切片将被标记。 </li><li>复制数据表与总α-突触核蛋白的表面，夹杂物的数量和平均粒径。粘贴到Excel电子表格或其他地方注明数据。 </li><li>返回斐济，并找到先前选定的切片中的β-微管蛋白通道，并选择它。 </li><li>选择神经节区域，然后单击分析，然后选择测量。另一个表中会出现显示总表面的神经节。 </li><li>从表中提取数据到Excel工作表的α-突触核蛋白的测量附近或其他地方将它复制下来。 </li><li>使用Excel，划分由神经节表面的α-突触核蛋白测量不同的参数，以获得总α-突触核蛋白的表面和包容的节大小归。 </li></ol> 4）代表性的成果当灌胃给药协议是正确执行动物的不便是微乎其微的。治疗这种鱼藤酮的浓度，使没有血液中的鱼藤酮水平上的ENS当地的鱼藤酮作用，并没有抑制肌肉或脑线粒体复合物，我什至1.5个月的治疗后（见图1）。如果图像分析是正确执行严格的分析的α-突触核蛋白的格局应该是可能的。它提供可靠的数据，对α-突触核蛋白（总表面积），α-突触核蛋白在细胞模式（夹杂物的数量）和存在的α- synuclein的夹杂物（包含大小）（图2）的总金额。 <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="图1" /> 图1。汽车没有检测鱼藤酮在血液或中枢神经系统功能障碍在鱼藤酮治疗的小鼠。 一，标准（50毫微克/毫升），并从20，10和5毫克/公斤治疗的小鼠的大脑样品的色谱图，B和C，血液中的鱼藤酮的水平（B）和CNS（三）量化，B，血药浓度1治疗后，2日和3小时。小鼠被分为三组（N = 3），2.5，5，10和20毫克/公斤鱼藤酮治疗组（n = 3），300μL的血液中提取1，2和3小时后，鱼藤酮管理和汇集为高效液相色谱法分析C，小鼠治疗一个星期一次5（N = 3），10（N = 3）和20天（N = 1）毫克/公斤的鱼藤酮，脑和脑干中提取1和2小时后末次给药和高效液相色谱分析D，线粒体复合物我1.5月治疗的小鼠的肌肉和大脑样本中的活动准备。 <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="图2" /> 图2。本地管理的鱼藤酮诱导α-突触核蛋白磷酸化，积累和聚集在ENS的神经节胶质细胞增生（比例尺20 微米），A，B，C，抗βIII-微管蛋白，α-突触核蛋白和十二指肠的DAPI染色（二）和回肠（ A，C）节。 在 B箭，1.5个月的治疗诱导增加α-突触核蛋白肠神经系统神经节内的点状模式相比，3个月控制（ 一 ） 。箭，治疗3个月诱导形成较大的α- synuclein的夹杂物（O&gt; 6 微米 ） 在 C，D，免疫荧光染色使用抗-α-突触核蛋白，硫黄素小号和DAPI染色。箭在 D中，只有3个月治疗的小鼠表明这些较大的α-突触核蛋白积累聚合，E，E&#39;，发送“，图像分析定量步骤，E，例如使用的图像显示一个ENS的神经节的量化阿尔法synuclein的免疫染色对显示地 ，定量实验显示在不同的夹杂物。E“，例如图像分析协议后获得的图像，E”，一些原来的夹杂物（绿色）和执行协议（黄线）后获得的代表性之间的重叠。 交流是使用自动分割和基于熵的阈值方法。单星号&lt;0.05，双星号，P &lt;0.01。楼 ，每一列代表总α- synuclein的表面/节表面，G，每一列代表总数的α- synuclein的夹杂物/节表面。所有图表显示平均± SEM

<ol>
	<li>Braak, H., de Vos, R. A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. R., de Vos, R. A., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+lesions+in+dorsal+horn+layer+I,+involvement+of+parasympathetic+and+sympathetic+pre-+and+postganglionic+neurons.">Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre- and postganglionic neurons.</a> Acta Neuropathol. 113, 421-429 (2007).</li><li>Braak, H., Ghebremedhin, E., Rub, U., Bratzke, H., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+in+the+development+of+Parkinson's+disease-related+pathology.">Stages in the development of Parkinson's disease-related pathology.</a> Cell Tissue Res. 318, 121-134 (2004).</li><li>Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., Richardson, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+toxicity+in+rotenone+models+of+Parkinson's+disease.">Mechanism of toxicity in rotenone models of Parkinson's disease.</a> J Neurosci. 23, 10756-10764 (2003).</li><li>Bove, J., Prou, D., Perier, C., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-induced+models+of+Parkinson's+disease.">Toxin-induced models of Parkinson's disease.</a> NeuroRx. 2, 484-494 (2005).</li><li>Inden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+of+mouse+nigrostriatal+dopaminergic+system+induced+by+repeated+oral+administration+of+rotenone+is+prevented+by+4-phenylbutyrate,+a+chemical+chaperone.">Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone.</a> J Neurochem. 101, 1491-1504 (2007).</li></ol>

旧金山泛Montojo为这种动物模型（申请号厘/ EP 2009/005688）的专利申请。

灌胃给药先前已执行12。然而，因登的手稿，鱼藤酮管理溶解在0.5％的羧甲基纤维素钠（CMSS）单。鱼藤酮是一种高liphophylic物质。因此，鱼藤酮不能被溶解在0.5％的CMSS单独会沉淀，如果这样做的。氯仿的使用创建一个均匀分布的鱼藤酮悬浮，避免沉淀。此外，由于在CMSS农药的浓度较高，管理解决方案的最终体积也明显下降。因此，减少并发症的管理。 我们建议使用?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
</tr>
<tr>
<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
</tr>
<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
</tr>
<tr>
<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FIJI</td>
<td>&nbsp;</td>
<td>http://pacific.mpi-cbg.de</td>
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oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

在帕金森氏病（PD）患者中，相关的病理如下特征的模式，特别是肠神经系统 （ENS）1,2，嗅球（OB），迷走“（DMV）3的背运动核，中间外侧脊髓4和黑质核，提供4,5疾病的神经病理分期的基础。的ENS和OB的最暴露的神经结构，首当其冲受到影响的。有趣的是，PD已经涉及到农药暴露6-8。在这里，我们详细显示在我们以前的研究9中使用的两种方法。为了分析的ENS本地的鱼藤酮的影响，我们使用一岁C57/BL6小鼠灌胃管理鱼藤酮。鱼藤酮是一种广泛使用的农药，强烈抑制线粒体复合物I 10。它是高度lipophylic和11在胃肠道吸收不良。我们的研究结果表明，5毫克/公斤鱼藤酮的管理并没有抑制线粒体复合物我的肌肉或大脑的活动。因此，这表明，我们的管理方法，鱼藤酮没有跨hepatoportal系统，是单纯的ENS行事。在这里，我们显示的方法来管理农药使用灌胃和图像分析协议，用来分析在α-突触核蛋白在ENS的积累农药的影响。第一部分显示了一个方法，使所需的精确浓度的农药（鱼藤酮）灌胃。第二种方法演示半自动图像分析协议分析α-突触核蛋白在ENS的积累，使用图像分析软件。

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

帕金森氏病已经涉及到农药的暴露。在这里，我们显示一个方法来提供所需浓度和方法来分析在α- synuclein的积累，在肠神经系统的影响农药的使用胃管。

鱼藤酮灌胃和图像分析α- synuclein的夹杂物在肠道神经系统的一个使用口服

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

oral-administration-rotenone-using-gavage-image-analysis-alpha

Research

JoVE Journal

Neuroscience

16.8K 次观看.  Technische Universität Dresden. 帕金森氏病已经涉及到农药的暴露。在这里，我们显示一个方法来提供所需浓度和方法来分析在α- synuclein的积累，在肠神经系统的影响农药的使用胃管。

视频: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

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 ...

科研

神经科学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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 ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...