我们想感谢Jeffrey罗斯坦提供BAC GLT1的绿色荧光蛋白的小鼠和GLT1抗体; Tufts中心神经科学研究所（NIH P30 NS047243 PI，罗布·杰克逊）提供有价值的核心设备，招聘新教师津贴（NIH P30 5P30NS069254-02 PI，菲尔·海登）在塔夫茨大学神经科学系。

1。大会的微流控文化厅（MCP） <ol><li> MCP（ 图1）是开放的腔室隔间培养不同类型的单元4设计。它典型地具有被连接的两个隔室，通过中央通道（直径为3微米）。大会MCP玻璃底的菜肴是必须的为编制文化和后续的图像分析。 </li><li>首先，外套的无菌玻璃有底菜肴用多聚鸟氨酸（Sigma-Aldrich公司，1毫克/毫升）溶解在四硼酸钠（Sigma-Aldrich公司，10mM的pH为8.5）中，并在37℃下孵育过夜</li><li>翌日，镀膜玻璃有底菜肴与DDH 2 O洗涤三次，并干燥无菌通风柜下。然后玻璃底船菜肴进一步涂覆与层粘连蛋白（Sigma-Aldrich公司，1毫克/毫升），并在UV光下干燥1小时。涂层菜已经准备好使用，也可以使用储存在-20°C至。这些涂重新所需的电镀神经元和神经胶质细胞的共培养。 </li><li>为了组装的文化平台，微流体文化平台被放置在顶部的涂覆的玻璃底菜肴与中央连接通道，在其底侧，形成一个紧密的密封。正常神经元或星形胶质细胞的培养基中加入（从细胞电镀区域）的两侧（ 图1）上到组装的MCP和在37℃下温育2-4小时，以确保有没有MCP和玻璃之间的泄漏底菜。我们已经测试了镀槽前的介质，以确保没有泄漏。组装MCP上的玻璃底的菜应新鲜配制，使用前。 </li></ol> 2。神经细胞的培养和诱导的神经元轴突在组装MCP的制备<ol><li>从胚胎14-16日龄小鼠的大脑皮质神经元细胞培养新鲜烹制的。神经元的培养基中，2％B27 neurobasa neurobasal培养基组成升补充，通过添加1％的100倍GlutaMAX，和1％青霉素 - 链霉素，2mM谷氨酰胺。小鼠大脑的解剖，在解剖显微镜下，从皮质脑膜。然后将组织剁碎用剃刀片，以增加的表面积暴露于胰蛋白酶，使小的组织块。胰酶消化组织块（Sigma-Aldrich公司0.05％胰蛋白酶），在37℃水浴10分钟，然后解离用火抛光的巴斯德吸管轻轻研磨。通过70微米的过滤器过滤，收集游离细胞明显的神经元细胞悬液。 </li><li>镀组装MCP的右侧（ 图1）上的细胞上的镀部分的神经元（2-3×10 6个 /毫升，150微升），使神经元可以附加在单元格中的保留区（ 图1）。新鲜制备的神经元与神经元电镀介质中，其中补充了5％牛胎儿血清（Hyclone）到稳压铺板ŕ神经元的培养基。因为只有附着在细胞保持区域的神经元，能够生长的轴突成通过中央通道的另一侧的组装MCP，板高密度的神经元细胞中电镀面积是重要的，以确保足够的神经元连接到细胞保留区。是在图2A中所示的神经元的轴突的高密度的一位代表图像。 </li><li>翌日，神经元的镀介质代替由经常的神经元的培养基。改变培养基，通过仔细地吸镀细胞的介质从腔室的面积，但不是从组装MCP细胞保留区，以避免在单元格中的保留区和中央连接通道的气泡。气泡会严重抑制轴突生长和后来进入中央通道在MCP。胶质细胞源性神经营养因子（GDNF，10〜20纳克/毫升）的另一侧（左侧）上也被加入在同一天的腔室，从组装MCP（ 图2A）通过中央通道的神经元的侧视图和横促进轴突生长5诱导。相当不GDNF的情况下，也观察到自发的神经轴突生长的神经元侧和交叉通过中央通道的组装MCP。通常可观察到轴突进入中央通道电镀后的2-3天。轴突一旦进入中央通道，它们通常在一天之内的另一侧输入。轴突通常是完整的至少一个星期后，进入另一侧。 </li></ol> 3。增加，建立条块分割的共培养系统培养的星形胶质细胞MCP 主要P1-3鼠标小狗大脑星形胶质细胞培养制备。脑分离程序是类似上述的神经元细胞的分离过程。星形胶质细胞，接种于10 cm培养皿中预涂用多聚鸟氨酸（Sigma-Aldrich公司，1毫克/毫升）。星形胶质细胞培养基（DMEM，10％胎牛血清，1％青霉素，链霉素），未来两天每天更换，以清除污物。在那之后，将培养基改变每三天。 <ol><li>星形胶质细胞成为90％汇合形成单层后7天。汇合的星形胶质细胞用胰蛋白酶消化和再悬浮的星形胶质细胞（1×10 6个 / ml）的150微升的重新组装MCP左侧进入细胞电镀面积镀覆时轴突即将进入或刚才订立的左侧组装的MCP。星形胶质细胞，通常接种后4-5天的神经元镀。的GDNF首先被除去之前，星形胶质细胞的再镀到左侧的MCP。附着在细胞中保留区再镀敷的星形胶质细胞，GFAP的免疫染色（ 图1）所示。只有很少的两侧腔室之间流动的介质（通过荧光ð确定是）被发现在MCP，如前所示4,6。 </li><li>星形胶质细胞成为90％汇合形成单层后7天。汇合的星形胶质细胞用胰蛋白酶消化和150微升的重新悬浮的星形胶质细胞（1×10 6个 / ml）被重新组装MCP左侧进入细胞电镀面积镀覆时轴突即将进入或刚订立左侧侧的组装MCP。星形胶质细胞，通常接种后4-5天的神经元镀。的GDNF首先被除去之前，星形胶质细胞的再镀到左侧的MCP。附着在细胞中保留区再镀敷的星形胶质细胞，GFAP的免疫染色（ 图1）所示。介质腔室的两侧之间只有最小流速（由荧光染料确定）被发现在MCP，如前所示4,6。 </li><li>后重新电镀的星形胶质细胞，轴突进入组装MCP左侧直接与ASTR交互ocytes通过直接的轴突接触或释放可溶性因子。免疫组织化学染色进行可视化的星形胶质细胞的质膜谷氨酸转运GLT1βIII-微管蛋白和神经元的轴突和星形胶质细胞之间的相互作用，如在图2D-F所示。 </li></ol>

<ol>
	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. D., Stevens-Graham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+neuron-glia+communication.">New insights into neuron-glia communication.</a> Science. 298, 556-562 (2002).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature. 1, 2128-2136 (2006).</li><li>Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuromuscular+synapse+development+by+glial+cell+line-derived+neurotrophic+factor+and+neurturin.">Regulation of neuromuscular synapse development by glial cell line-derived neurotrophic factor and neurturin.</a> The Journal of biological chemistry. 277, 10614-10625 (2002).</li><li>Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+astroglial+excitatory+neurotransmitter+transporter+GLT1.">Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.</a> Neuron. 61, 880-894 (2009).</li><li>Regan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+promoter+activity+reveal+a+differential+expression+and+physiology+of+glutamate+transporters+by+glia+in+the+developing+and+mature+CNS.">Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS.</a> The Journal of neuroscience. 27, 6607-6619 (2007).</li><li>Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+glutamate+transporter+subtype+expression+in+astrocytes.">Neuronal regulation of glutamate transporter subtype expression in astrocytes.</a> The Journal of neuroscience. 17, 932-940 (1997).</li><li>Schlag, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+glial+Na+-dependent+glutamate+transporters+by+cyclic+AMP+analogs+and+neurons.">Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.</a> Molecular pharmacology. 53, 355-369 (1998).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> Journal of immunological. 300, 32-46 (2005).</li><li>Espinosa-Jeffrey, A., Wakeman, D. R., Kim, S. U., Snyder, E. Y., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+system+for+rodent+and+human+oligodendrocyte+specification,+lineage+progression,+and+maturation.">Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation.</a> Current protocols in stem cell biology. Chapter 2, (2009).</li><li>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=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Debanne, D., Rama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+shape+axonal+signaling.">Astrocytes shape axonal signaling.</a> Science signaling. 4, pe11 (2011).</li></ol>

没有利益冲突的声明。

MCP基于神经元和星形胶质细胞共培养体系可以详细的神经元，星形胶质细胞信号转导通路的解剖允许唯一的轴突通过中央通道，并与星形胶质细胞。此共培养体系可以方便地设置与传统的神经元，星形胶质细胞的培养程序。我们也采用的绿色荧光蛋白记者展示轴突取决于GLT1的启动子活性在星形胶质细胞共培养系统的实际应用。 这种MCP基于神经元和星形胶质细胞共培养体系提...

<table border="1"><tbody><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070.03 </td><td>电镀神经元神经元cutlure培养基</td></tr><tr><td>胎牛血清</td><td> Sigma-Aldrich公司</td><td> F4135 </td><td>星形胶质细胞培养基</td></tr><tr><td>胶质细胞源性神经因子</td><td> R＆D系统</td><td> 212-GD </td><td> 10-20毫微克/毫升侧腔的神经元</td></tr><tr><td> Dulbecco改良Eagle培养基高糖</td><td> Sigma-Aldrich公司</td><td> 11995 </td><td></td></tr><tr><td> 70毫米电池过滤器， </td><td> BD猎鹰</td><td> 352350 </td><td></td></tr><tr><td>无菌玻璃底菜</td><td> MatTek公司</td><td></td><td></td></tr><tr><td>微流控文化平台</td><td> XONA微流体有限责任公司</td><td> SND150 </td><td></td></tr><tr><td> 6个孔的培养板</TD&gt; <td>蜂星</td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> 神经元培养基 <ul><li> Neurobasal培养基</li><li> 2％B27的Neurobasal补充</li><li>通过添加1％的100倍GlutaMAX 2mM的谷氨酸</li><li> 1％青霉素 - 链霉素</li></ul></td></tr><tr><td></td><td></td><td></td><td> 的镀敷细胞的神经元的培养基 <ul><li> Neurobasal培养基</li><li> 2％B27的Neurobasal补充</li><li>通过添加1％的100倍GlutaMAX 2mM的谷氨酸</li><li> 1％青霉素 - 链霉素</li><li> 5％胎牛血清SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> 星形胶质细胞培养基 <ul><li> Dulbecco改良EAGLE中高血糖</li><li> 10％胎牛血清F4135 </li><li> 1％青霉素 - 链霉素</li></ul></td></tr><tr><td></td><td></td><td></td><td> 表1中。材料中使用的微流体培养基于平台的神经元轴突和神经胶质细胞的共培养系统。 </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

适当的神经元，神经胶质细胞相互作用的中枢神经系统（CNS）的生理功能是至关重要的。这种双向通信老练介导的神经元和神经胶质细胞1,2之间通过特定的信号转导通路。这些信号通路的识别和表征如何神经元的神经胶质细胞相互作用的形状中枢神经系统生理学的理解是必不可少的。在此之前，神经元和神经胶质细胞混合培养已被广泛用于测试和表征神经元和神经胶质细胞之间的信号传导通路。我们已经从这些制剂及其他体内的工具，但是，已经表明，神经元和神经胶质细胞相互之间的信号往往发生在特定的车厢内神经元（ 即轴突，树突或胞体）3。这使得重要的是要开发一种新的文化系统，让神经车厢分离，专门研究神经胶质细胞和神经之间的相互作用NAL轴突/树突。此外，以往的混合培养系统是不能够分化的可溶性因子和神经元和神经胶质细胞之间的直接膜接触信号。此外，大量的神经元和神经胶质细胞，在传统的共培养体系缺乏必要的分辨率，观察到一个单一的轴突和神经胶质细胞之间的相互作用。 在这项研究中，我们描述了一种新的轴突和神经胶质细胞共培养系统与使用平台（MCP）的微流体文化。在此共同培养系统中，神经元和神经胶质细胞培养在两个单独的腔室，通过连接多个中央通道。在这个微文化平台，神经元的过程（特别是轴突）可以通过中央通道进入的胶质。结合强大的荧光蛋白标记，该系统可以直接检查/树突状轴突和胶质细胞相互作用的信号转导通路之间，这样的小号轴突神经胶质细胞，神经胶质细胞介导的​​受体贩运神经元的终端，和神经胶质细胞介导的​​轴突生长介导的转录调控。的窄直径的腔室，也显着到胶质室禁止神经元的富集培养基的流，促进轴突/树突和胶质表面之间的直接膜 - 蛋白质相互作用的探测。

轴突诱导GLT1的启动子活性在星形胶质细胞延时成像分析隔间的神经元和星形胶质细胞的共培养系统允许的神经元的过程，尤其是在轴突，以选择性地与星形胶质细胞相互作用。不同类型的轴突神经胶质细胞相互作用的成功建立在组装MCP的轴突和星形胶质细胞（或其他神经胶质细胞）共培养后，可以研究，如轴突诱导活化的星形胶质细胞的基因启动子活性，星形胶质细胞诱导的?...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

本研究介绍了一种新的神经元轴突和神经胶质细胞共培养（ASTRO）平台的程序。在此共培养体系中的一个单一的轴突（及单一的神经胶质细胞）之间的直接相互作用，操纵变得可行，允许机械分析的相互神经胶质信令。

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K 次观看.  Tufts University. 本研究介绍了一种新的神经元轴突和神经胶质细胞共培养（ASTRO）平台的程序。在此共培养体系中的一个单一的轴突（及单一的神经胶质细胞）之间的直接相互作用，操纵变得可行，允许机械分析的相互神经胶质信令。

视频: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

科研

神经科学

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

微流控采用共培养开发设备牙胚支配分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

脑回路非侵入性断开的靶向神经元损伤

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...