这项工作是由美国国家精神卫生研究所和美国国家科学基金会的支持。许多有益的讨论和建设性的批评，的Olivier Paugois的专家鱼的饲养的西伯实验室成员的特别感谢。

1。准备显微注射<ol><li>准备显微注射针拉毛细管萨特仪器针拔出器。 </li><li>负载微量注射针用荧光染料（FITC-葡聚糖）。 </li><li>显微操作显微注射设备上安装针。 </li><li>使用产钳，宽度约2微米，小心地打破显微注射针，但是，这将取决于你的微量注射器设置。对于我们的显微注射针，这对应于从不会弯曲的前端的针的第一区域。 </li><li>测量在油中的液滴大小，调整喷射时间和压力，从​​而使每次注射提供1 NL。哈佛设备微量注射器的示例设置为：P余额= 1.4，P出= 1.4磅磅，P = 22.9的PSI，P清晰= 67.8磅注入与注射时间为0.4到0.7秒。我们使用这些设置的针的直径大约是2微米。不过，设置特定的微量注射器，并改变一个ccording针直径。 </li></ol> 2。准备胚胎<ol><li>外套2菜肴与在水中的1％琼脂糖的每个条件，用1-200微升的移液管尖端到琼脂糖戳孔，和除去琼脂糖插头。填写菜与胚胎媒体。 </li><li>使用钳，dechorionate的胚胎，在立体显微镜下为18 HPF或以上。胚胎都在上演金梅尔等。11。 </li><li> dechorionated胚胎转移到第一琼脂糖凝胶涂层的菜。 </li><li>若要麻醉胚胎，三卡因（0.1毫克/毫升）添加到培养皿中，直到胚胎停止移动（韦斯特12根据）。 </li></ol> 3。脑室注射<ol><li>东方胚胎，所以你看他们的背侧把尾部的胚胎成孔。如果你的微操作机器人是在右边，然后将胚胎，使前脑的左侧和后脑的权利。 </li><li>在无线定位针目标点的后脑心室。 </li><li>小心刺破屋顶板的后脑心室的蛋黄（ 图1A），千万不要去通过深入的大脑。 </li><li>确保染料填充的整个长度的脑室入脑室注射1-2 NL的荧光染料。 </li><li>第二琼脂糖凝胶涂层菜充满胚胎媒体的移植胚胎，重新麻醉在2.4。 </li><li>立刻开始，如在第4所述的成像，以获得时间零点图像。 </li></ol> 4。成像<ol><li>东方胚胎的尾巴孔与3.1。 </li><li>在解剖显微镜使用一个明场背图像传输和荧光灯。不同的胚胎成像之间的放大倍率保持恒定。这样就可以直接使用图像J（5.2-6）进行分析比较。 </li><li>不动，胚胎，显微镜或菜，取相应的荧光图像。 </li><li>重复为每个胚胎在期望的时间点。 </li></ol> 5。量化染料运动<ol><li>合并明场和荧光图像在Photoshop如前所述由Gutzman和西伯10。 </li><li>测量距离的染料Image J软件可在<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>前移动。 </li><li>打开合并后的文件图像J和使用直线工具绘制一条线从的前脑铰链点，染前在10〜20°角从神经上（ 图1A）。这一区域被选择，因为它是第一和最引人注目的站点的染料泄漏出来的野生型神经上皮。 </li><li>选择测量工具来计算线路长度。 </li><li>每个时间点重复上述步骤。 </li><li>计算净距离动议染料前端减去从其他时间点t = 0时的距离随着时间的推移。 </li><li>剧情上图。 </li></ol> 6。代表性的成果在的的神经上皮渗透性测定使用野生型胚胎获得的结果的一个例子示于图1B-D。为了准确区分磁导率，它是有用的，以测试染料与不同分子weightsto识别的尺寸，是在野生型或控制胚胎（ 图2），只是稍微漏。这允许鉴定遗传突变体或环境条件下，无论是增加或减少的磁导率（ 图1D，分别为绿色和红色线）。对于24 HPF斑马鱼神经上皮，70 kDa的FITC葡聚糖泄漏慢慢超过2小时，而2000 kDa的不和10kDa几乎立刻泄漏出来。因此，70 kDa的分子量是理想的，以确定条件，增加和减少神经上皮通透性。 ，如果针想念的心腔，荧光瓦特外脑病出现在t = 0（一个例子Gutzman和西伯，2009年10月 ）。由于注入的染料没有最初载大脑内的，并没有明确作出结论有关运动的染料和渗透率的neuropeithelium的，应该被丢弃这些胚胎。 最后，如​​果的胚胎有小的脑室或非膨胀的脑室，预喷射之前可以先完成荧光染料注射用生理盐水溶液的心室。这膨胀心室发出后续的可视化时更容易与荧光染料注入脑室。必须进行适当的控制，以确定是否注射生理盐水，扰乱了正常的神经管发育。 <img alt="图1" src="/files/ftp_upload/4242/4242fig1.jpg" /> 图1。时间过程的不同分子量的染料 （A）的实验图。第一，荧光染料注入脑室。 X =用于注射针的位置。下一步的背拍摄的图像随着时间的推移。最后，移动的距离测量铰接点（由一个红色的线）表示的染料从前方的前脑。 （BC）22 HPF（T = 0分，B）和24 HPF（T = 120分钟，C）明场和荧光背的影像合并。白线表示的染料前脑脑室前部的距离。 （D）假设的样品渗透率数据。蓝=野生型或控制的，红色样品的渗透率下降的相对控制，绿色=采样与通透性增加，相对控制。 <img alt="图2" src="/files/ftp_upload/4242/4242fig2.jpg" /> 图2。测量神经上皮渗透性不同分子量的染料 （AE）背合并的明视野图像和荧光图像22高倍视野野生型胚胎在t = 0分钟后，用FITC-D注射EXTRAN以下分子量：10 kDa的（A），（B）40 kDa的，70 kDa的（C），500 kDa的（D）和2,000 kDa的（E）。 （A&#39;-E&#39;）同胚（AE），在t = 120分钟，24 hpf时。前方左。 F =前脑，M =中脑，：H =后脑的。 Asterisk的耳朵。

<ol>
	<li>Harrington, M. J., Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+neurulation:+first+impressions+do+not+count.">Comparative analysis of neurulation: first impressions do not count.</a> Mol. Reprod. Dev. 76, 954-965 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+vertebrate+neurulation+and+a+re-evaluation+of+teleost+neural+tube+formation.">Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation.</a> Mech. Dev. 121, 1189-1197 (2004).</li><li>Salehi, Z., Mashayekhi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cerebrospinal+fluid+on+neural+cell+survival+in+the+developing+chick+cerebral+cortex:+an+in+vivo+study.">The role of cerebrospinal fluid on neural cell survival in the developing chick cerebral cortex: an in vivo study.</a> Eur. J. Neurol. 13, 760-764 (2006).</li><li>Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryonic+brain+development+in+rats+requires+the+trophic+influence+of+cerebrospinal+fluid.">Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid.</a> Int. J. Dev. Neurosci. 27, 733-740 (2009).</li><li>Lehtinen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebrospinal+fluid+provides+a+proliferative+niche+for+neural+progenitor+cells.">The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.</a> Neuron. 69, 893-905 (2011).</li><li>Gato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cerebrospinal+fluid+regulates+neuroepithelial+survival,+proliferation,+and+neurogenesis+in+chick+embryos.">Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos.</a> Anat. Rec. A. Discov. Mol. Cell Evol. Biol. 284, 475-484 (2005).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Totally+tubular:+the+mystery+behind+function+and+origin+of+the+brain+ventricular+system.">Totally tubular: the mystery behind function and origin of the brain ventricular system.</a> Bioessays. 31, 446-458 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+neuroepithelial+barrier+by+Claudin5a+is+essential+for+zebrafish+brain+ventricular+lumen+expansion.">Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1425-1430 (2010).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+brain+ventricle+injection.">Zebrafish brain ventricle injection.</a> J. Vis. Exp. , (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M., Sprague, J., Doerry, E., Douglas, S., Grp, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Information+Network+(ZFIN):+a+resource+for+genetic,+genomic+and+developmental+research.">The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.</a> Nucleic Acids Research. 29, 87-90 (2001).</li></ol>

没有利益冲突的声明。

我们证明了量化的活体胚胎斑马鱼脑作为给定的分子量的确定注入的染料的渗透性的能力。我们的观察斑马鱼胚胎神经上皮差异透性的染料不同的分子量，表明了染料的移动经由间通透性。但是，我们不能排除的可能性的跨细胞所观察到的磁导率的贡献。这种技术可以应用于任何其他的管状结构，只要管的内侧和外侧的，可以看出，可以注射和管腔。然而，确定了理想的分子量为其他器官将需要?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>葡聚糖，荧光素，阴离子，赖氨酸绝不破裂</td><td> Invitrogen公司</td><td> D7136，D7137，D1822，D1820，D1845 </td></tr><tr><td>三卡因粉</td><td>西格玛</td><td> A5040 </td></tr><tr><td>毛细管</td><td> FHC公司</td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

脑脑室系统脊椎动物之间是保守的，并且是由一系列互连的空腔称为脑脑室，大脑发育的早期阶段期间形成的，并保持整个动物的生命。被发现在脊椎动物脑脑室系统和心室神经管形成后，当中央管腔内充满脑脊液（CSF）1,2。 CSF是一种富含蛋白质的液体，是必不可少的正常的大脑发育和功能3-6。 在斑马鱼中，脑室通胀开始于受精后约18小时（HPF），神经管闭合后。多个进程相关联的，与脑室形成，包括一个神经上皮形成，形成紧密连接，调节渗透性和脑脊液生产。我们发现，中Na，K-ATP酶活性，影响所有这些过程所需的脑室通胀ES 7.8，而紧密连接蛋白5A是必要的紧密连接形成9。此外，我们发现，“放松”的胚胎神经上皮，通过抑制肌球蛋白，与脑室通胀。 探讨在斑马鱼脑室通胀调节的透气性，我们开发了一个心室染料保持试验。此方法使用在活的斑马鱼胚胎，在我们的实验室10以前开发的技术，以荧光标记的脑脊液脑室注射。作为荧光染料穿过脑室和神经上皮胚胎，然后随着时间的推移成像。染料前端的距离移动远离基底（非管腔）侧，随着时间的推移的神经上皮被量化和是神经上皮渗透性的量度（ 图1）。我们观察到，70 kDa的和更小的染料会移动通过神经上皮和可detecte的D 24高倍视野（ 图2）以外的胚胎斑马鱼脑。 该染料保留检测可以用于分析在各种不同的遗传背景中的神经上皮通透性，在开发过程中在不同的时间，和环境扰动后。这也可能是有用的检查病​​理积累的CSF。总体而言，这种技术可以让调查人员分析的作用和调节的透气性，在开发过程中和疾病。

Watch this Scientific Journal Video about An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium at JoVE.com

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

我们描述了现场的整个动物的胚胎斑马鱼脑通透性的定量测量。该技术分析神经管的管腔内保留脑脊液和不同分子量的分子的能力，和它们的运动量化满分心室。此方法是非常有用的用于确定在上皮通透性和成熟发展和疾病过程中的差异。

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-assay-for-permeability-of-the-zebrafish-embryonic-neuroepithelium

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Massachusetts Institute of Technology. 我们描述了现场的整个动物的胚胎斑马鱼脑通透性的定量测量。该技术分析神经管的管腔内保留脑脊液和不同分子量的分子的能力，和它们的运动量化满分心室。此方法是非常有用的用于确定在上皮通透性和成熟发展和疾病过程中的差异。

视频: An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

科研

神经科学

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

手动排水的斑马鱼胚胎脑室

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录：神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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.