Puncta分析仪插入图像J的巴里Wark（地址：Physion咨询）写在本A.巴雷斯实验室（斯坦福大学）。 资金; <ul><li> Alfred P. Sloan基金会</li><li>以斯帖A.和约瑟夫Klingenstein基金公司</li><li>广泛的生物医学研究基金会</li><li>治疗亨廷顿氏病倡议</li></ul>

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	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

上文所述突触检测是基于在我们的实验目标，其中主要集中于视网膜神经节细胞兴奋的预测中，无论是在纯化的文化，或在大脑部分。我们提供了一个参考表中列出的标签（见表1）兴奋性突触的抗体，工作。 这种突触的检测，可适应量化任何神经元的人口或任何其他的突触亚型，这是有选择性的前和突触后标记的突触数量。例如，突触实验描述在这里可以应用到研究的GABA - ...

<table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 抗原 </td><td> 物种 </td><td> 单克隆/多克隆 </td><td> 供应商 </td><td> 产品编号 </td><td> 稀释 </td><td> 文化工程 </td><td> 可在第 </td></tr><tr><td rowspan="10">突触前</td><td>突触</td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 106004 </td><td> 1:750 </td><td> Ÿ </td><td> ND </td></tr><tr><td>突触</td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 106001 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>巴松管</td><td>鼠标</td><td>单克隆抗体</td><td>检测设计</td><td> VAM - PS003F </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>巴松管</td><td>豚鼠</td><td>多克隆</td><td>突触系统</td><td> 141004 </td><td> 1:1000 </td><td> Ÿ </td><td> ND </td></tr><tr><td>突触结合蛋白1 </td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 105002 </td><td> 1:750 </td><td> Ÿ </td><td> ñ </td></tr><tr><td> Synaptobrevin 2 （C1.69.1） </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 104211 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>突触 （C1.7.2） </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 101011 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> VGlut1 </td><td>鼠标</td><td>单克隆抗体</td><td> Millipore公司</td><td> MAB5502 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td> VGlut1 </td><td>豚鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB5905 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td> VGlut2 </td><td>豚鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB2251 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td rowspan="6">突触后</td><td> PSD - 95 （6G6 - 1C9克隆） </td><td>鼠标</td><td>单克隆抗体</td><td>亲和生物试剂</td><td> MA1 - 045 </td><td> 1:750 </td><td> Ÿ </td><td> ñ </td></tr><tr><td> PSD - 95 </td><td>兔</td><td>多克隆</td><td> Zymed公司</td><td> 51-6900 </td><td> 1:500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td>荷马</td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 160011 </td><td> 1:500 </td><td> Ÿ </td><td> ND </td></tr><tr><td>荷马</td><td>大鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB5875 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> Gephyrin </td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 147003 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> Gephyrin </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 147 </td><td> 1:200 </td><td> Ÿ </td><td> Ÿ </td></tr></tbody></table> 表1：好，我们已经成功地利用我们的突触检测前和突触后的标记列表的例子。要知道，这不是一个详尽的列表所有可用的标记。 Y =是，N =否，ND =未确定。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫。号 </td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 20012-027 </td></tr><tr><td>聚- D -赖氨酸</td><td>西格玛</td><td> P6407 </td></tr><tr><td>层粘连蛋白</td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td> TRITON X - 100 </td><td>罗氏诊断有限公司</td><td> 9002-93-1 </td></tr><tr><td>正常山羊血清</td><td> Gibco公司</td><td> 16210 </td></tr><tr><td> VectaShield用DAPI </td><td>媒介实验室</td><td> H - 1200 </td></tr><tr><td>华侨城</td><td>组织Tek的</td><td> 4583 </td></tr><tr><td>三基地（50毫米） </td><td>费舍尔</td><td> BP152 - 5 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A2153 </td></tr><tr><td> L -赖氨酸</td><td>西格玛</td><td> L - 1137 </td></tr><tr><td> 16％PFA解决方案</td><td>电子显微镜科学</td><td> 15711 </td></tr><tr><td>颗粒煤灰</td><td>电子显微镜科学</td><td> 19210 </td></tr><tr><td> 24孔培养板</td><td>鹘</td><td> 35-3047 </td></tr><tr><td>羊抗鼠Alexa的共轭抗体</td><td> Invitrogen公司</td><td> --- </td></tr><tr><td> 供应 </td><td> 公司 </td><td> 猫。号 </td></tr><tr><td> 12MM，0号盖玻片</td><td>卡尔赫克特有限公司</td><td> 1105209 </td></tr><tr><td> 1.5玻璃盖玻片（片） </td><td> VWR科学</td><td> 48393241 </td></tr><tr><td>载玻片</td><td> VWR科学</td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

在神经科学中最重要的目标之一是要了解分子线索指示突触形成的早期阶段。因此，它已成为当务之急，制定客观的方法来量化突触连接的变化。从样品固定开始，这个协议的细节如何量化无论是在分离的神经元文化和在脑切片，用免疫细胞化学突触的数量。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。这些colocalizations量化Puncta分析器（巴里Wark，可根据要求，c.eroglu @ cellbio.duke.edu书面）使用ImageJ分析软件平台下的一个插件。在本议定书中所描述的突触检测可应用于任何神经组织或文化的准备，你有选择性的前和突触后标记。这突触法是一种宝贵的的工具，可以被广泛利用在突触发展的研究。

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

该协议的细节，如何量化，在分离神经元的文化和突触数量在脑切片，用免疫细胞化学。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。

突触量化：基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

quantifying-synapses-an-immunocytochemistry-based-assay-to-quantify

Research

JoVE Journal

Neuroscience

35.4K 次观看.  Duke University. 该协议的细节，如何量化，在分离神经元的文化和突触数量在脑切片，用免疫细胞化学。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。

视频: Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

的斑马鱼胚胎神经上皮通透性的测定

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

视网膜原酸和皮质化突触功能的电生理学研究

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...