这项工作是从杰克逊实验室的启动资金支持。

1。准备鼠神经胶质细胞培养的神经元培养空调<ol><li>涂有胶原蛋白溶液（1:3稀释Purecol DDH 2 O中）的T75烧瓶。烧瓶一身正气，干通宵的组织文化引擎盖。 </li><li>夹层缓冲液（人工脑脊液：119 mM氯化钠，5 mM KCl中，1毫摩尔MgCl 2和30mM葡萄糖，25mM的HEPES，pH值7.4，无钙）和神经胶质细胞介质（DMEM培养液与10％胎牛血清，10单位/ ml青霉素，10 μg/ ml链霉素，和Glutamax）的制备和贮存在4℃下，直到准备使用。 </li><li>在当天的神经胶质细胞培养，神经胶质细胞介质加热在37°C水浴老鼠的大脑解剖前至少30分钟。小鼠胚胎或新生儿安乐死的快速斩首。在解剖显微镜下分离出脑皮层。 </li><li>解剖脑组织在木瓜蛋白酶溶液（100毫升木瓜蛋白酶和20μl1％DNA酶I在2毫升夹层缓冲液）在37℃下消化FOR 20分钟。 </li><li>木瓜蛋白酶消化后，脑组织用10ml预先温热的神经胶质细胞介质漂洗。新鲜的神经胶质细胞的介质（2毫升），然后加入到消化的脑组织，用火抛光的玻璃巴斯德吸管研磨和组织。 </li><li>新鲜的神经胶质细胞的介质（8毫升）中加入游离的组织。 A 70微米的细胞过滤器，用于过滤分离的组织悬液。然后将细胞接种于T75培养瓶（一般2-3个胚胎，种子T75烧瓶），置于37℃细胞培养孵化器。 </li><li>第二天，从T75烧瓶中的是媒体吸气。将烧瓶用10ml PBS漂洗。新鲜的神经胶质细胞介质（10-15毫升），然后加入到每个烧瓶中。 </li><li>在10-12天，神经胶质细胞在T75烧瓶中应该是融合。胶质细胞用PBS漂洗，和5毫升的0.05％胰蛋白酶被添加到每个的T75烧瓶中。 </li><li>胰蛋白酶在37℃下温育5分钟后，神经胶质细胞是从每个T75烧瓶中的解离和神经胶质细胞被分成两个不同的胶原蛋白涂覆的T75烧瓶。 </li><li>培养插入（Millipore公司PICM03050）的制备是通过将它们放置到6孔板中，涂层与胶原蛋白，并空气干燥过夜。神经胶质细胞介质（每次2毫升）置于下方插入每一种文化。 </li><li>胶质细胞用胰蛋白酶消化，并接种到培养插入从汇合的T75烧瓶（1的T75烧瓶中每6孔板中，每次2毫升插入）。这些神经胶质细胞培养插入将使用条件的培养的神经元。 </li></ol> 2。神经元培养<ol><li>神经细胞培养的盖玻片进行清洗浸泡在70％HNO 3，至少过夜。然后用盖玻片DDH 2 O的至少3倍，并且Isotemp烘箱中干燥（&gt; 200℃）中过夜。 </li><li>清洗的盖玻片放置在6孔板中，每孔的一个盖玻片。盖玻片都涂有聚-L-赖氨酸的水溶液（2毫升，0.1％在100mM硼酸，pH值= 8.5）中，在37℃培养箱中培养过夜。 </li><li> “聚-L-赖氨酸溶液的除去从盖玻片和盖玻片与蒸压DDH 2 O洗涤3次盖玻片然后，保温在37℃培养箱中培养过夜的电镀介质（补充有5％热灭活的马血清，10单位/ ml青霉素，10μg/ ml链霉素，Glutamax; 2％B27补充剂的Neurobasal添加到镀介质的当天使用过它）。 </li><li>翌日，除去镀敷介质和2毫升的新鲜电镀媒体与B-27被添加到各孔中，有一个玻璃盖玻片。 </li><li>怀孕的小鼠（E15-18）被安乐死与CO 2，和小鼠胚胎用断头安乐死。海马或纹状体解剖从脑组织中，并用冰冷的夹层缓冲区，用于培养的海马神经元或纹状体介质棘状神经元，分别放入15毫升锥形管。 </li><li>解剖脑组织用木瓜蛋白酶解离，在步骤1.4）和1.5）中所描述。 </li><li> Following解离，每个神经元的悬浮液中的细胞数用血细胞计数器计数。神经元，然后接种于每个盖玻片的6孔板中，每孔的密度为0.4×10 6个细胞，并且培养在37℃的培养箱中培养过夜（神经元是DIV 0）。 </li><li>为了制备用于调节的培养的神经元，神经胶质细胞培养插入的神经胶质细胞介质除去从神经胶质细胞培养插入和新鲜电镀介质中加入下面的插入物中的插入件和2毫升（2毫升）。 </li><li>第二天（DIV 1），与神经元的盖玻片置于下方的神经胶质细胞培养插入，在各孔中的一个盖玻片。 </li><li>神经元的输入与体积的一半新鲜预热的供给媒体（B-27），每3​​-4天，直到他们准备的转染和成像实验。 </li></ol> 3。转染<ol><li>神经细胞转染用脂质体2000。对于每一个神经细胞培养（每一种文化盖玻片），2微升使用脂质体2000和1微克DNA。没有补充新鲜的Neurobasal培养基被用来稀释脂质体和DNA。 </li><li> DNA与Lipofectamine混合后在室温下温育20分钟。 </li><li>下次从每个插入件内部的1毫升的媒体从每个神经胶质细胞培养插管的下方，和1毫升，将介质传送到一个​​锥形管。保存的介质和孵育在37℃，加入相同体积的送入介质+ B27此介质被用于转染后的神经元培养。 </li><li>脂质体/ DNA孵育20分钟之后，神经胶质细胞培养插入瞬间从6孔培养板中除去。 200μl的脂质体/ DNA混合物被添加到每个与神经元的盖玻片。神经胶质细胞培养插入各孔中，然后返回到上述的神经元。文化插入膜下的气泡的生成，应避免。神经元与脂质体/ DNA混合物在37℃下温育2-4小时。 </li><li>后日Ë2-4小时的潜伏期，神经胶质细胞中插入被删除了。与脂质体/ DNA混合物的培养基从各孔除去。步骤3.3）中保存的介质被添加到神经元（2毫升每孔）。神经胶质细胞培养插入返回到各孔，除去从每个插入的神经胶质细胞介质，并从步骤3.3）中的2毫升的介质加入到每个插入件。 </li><li>神经元培养24-72小时，让这些神经元表达的转基因前TIRF成像。 </li></ol> 4。 TIRF成像<ol><li>人工脑脊液（人工脑脊液：119 mM氯化钠，2.5mM的氯化钾，30mM葡萄糖，2mM的的CaCl 2，1毫摩尔的MgCl 2，25mM的HEPES，pH值= 7.4），将被用于活成像实验。我们的TIRF成像系统是自定义设置了100-mW的青色激光激发，电子倍增电荷耦合器件（EMCCD）相机的检测器。 </li><li>成像实验前，学联组织警告说，在37＆D例如，C水浴至少30分钟。实时成像的加热刀片被放置在显微镜载物台上，并加热刀片，激光，和摄像机的成像实验开始前至少30分钟温热。 </li><li>与转染的神经元的是，甲盖玻片活成像室组装成。预先温热的人工脑脊液被放置在被组装后的腔室的腔室，此步骤中，应尽可能快地完成。 </li><li>一旦被放置在腔室上的显微镜载物台的加热刀片，转染的神经元被确定视觉外延荧光成像模式下。 </li><li>健康转染后的神经元被识别（参照图1和图2），我们通常执行1分钟的光漂白剂，以消除在质膜上，预先存在pHluorin荧光在质膜上的pHluorin-受体具有非常高的荧光水平时第一次启动TIRF成像，干扰可视化的个人插件ertion事件。 </li><li>光漂白剂，EMCCD相机上的电子倍增器的增益设定是设定为最大，并进行记录，持续1分钟，在10赫兹。 </li><li>每个记录将包含600个图像。个别插入的事件都通过播放录音背面使用ImageJ。个人的插入手动使用ImageJ分析事件。 </li></ol> 5。代表性的成果一致的神经细胞培养的关键是成功的实时成像实验。 图1显示了小鼠海马神经元培养我们的协议从11 DIV息18。很明显，从图像的神经元建立了广泛的过程，而树突覆盖密集的树突棘，迹象表明，这些神经元在文化上是健康的。我们的培养方法也可用于其他类型的神经元在大脑。例如，我们最近使用的协议，以文化纹状体中等棘神经元在一项研究中，研究多巴胺D2受体（DRD2）插入在培养的小鼠纹状体的中等棘神经元9。 图2显示了小鼠纹状体中等棘神经元培养8 DIV使用我们的协议。我们还成功地进行这种类型的神经元中的superecliptic pHluorin标签DRD2s TIRF成像。 图3示出的表达pHluorin标签多巴胺D2（pH值-DRD2）在培养基中的多刺的pH-的DRD2插入在该神经元的神经元和可视化。 <img alt="图1" src="/files/ftp_upload/4450/4450fig1.jpg" /> 图1。小鼠海马文化的使用界面培养法。 DIV： 体外日，这一天被认为是文化DIV 0。从图像中，显而易见的是，在这种类型的文化之间的DIV 11和15 DI​​V成熟的海马神经元。在11和13的DIV，神经元树突与filipodia和未成熟棘覆盖。在DIV 1518，神经元树突覆盖着大刺，显示更成熟的神经元。左图：低倍率的图像在不同年龄的小鼠海马神经元。右图：高倍率的图像变焦在左侧面板中的神经元树突的神经元树突，树突棘在不同的年龄。 <img alt="图2" src="/files/ftp_upload/4450/4450fig2.jpg" /> 图2。鼠标纹状体中等多刺的神经元培养使用接口的培养方法。在图像的神经元在DIV 8。 <img alt="图3" src="/files/ftp_upload/4450/4450fig3.jpg" /> 图3：鼠标纹状体中多刺的神经元转染超黄道pHluorin标记多巴胺D2受体（PH-DRD2）。图片的左边是随时间推移记录的最大强度投影（600个图像，100毫秒每幅图像）。白色箭头指示个人水泡的pH-D插入RD2。此图片保留在xy尺寸的插入信息，但失去的时间信息。在右边的图像示出YT最大强度投影，在其中的XYT堆栈围绕y-轴旋转90度，并在每个x轴的线中的最大像素被投射到一个单一的y轴像素。此图像保留沿YT轴插入信息，但失去了x轴信息。

<ol>
	<li>Thomas, G. M., Hayashi, T., Chiu, S. L., Chen, C. M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylation+by+DHHC5/8+targets+GRIP1+to+dendritic+endosomes+to+regulate+AMPA-R+trafficking.">Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking.</a> Neuron. 73, 482-496 (2012).</li><li>Anggono, V., Huganir, R. L. <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+AMPA+receptor+trafficking+and+synaptic.">Regulation of AMPA receptor trafficking and synaptic.</a> Curr. Opin. Neurobiol. , (2012).</li><li>Makuch, L., Volk, L., Anggono, V., Johnson, R. C., Yu, Y., Duning, K., Kremerskothen, J., Xia, J., Takamiya, K., Huganir, R. L. <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+AMPA+receptor+function+by+the+human+memory-associated+gene+KIBRA.">Regulation of AMPA receptor function by the human memory-associated gene KIBRA.</a> Neuron. 71, 1022-1029 (2011).</li><li>Thomas, G. M., Lin, D. T., Nuriya, M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+bi-directional+regulation+of+AMPA+receptor+phosphorylation+and+trafficking+by+JNK.">Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK.</a> EMBO J. 27, 361-372 (2008).</li><li>Lin, D. T., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PICK1+and+phosphorylation+of+the+glutamate+receptor+2+(GluR2)+AMPA+receptor+subunit+regulates+GluR2+recycling+after+NMDA+receptor-induced+internalization.">PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization.</a> J. Neurosci. 27, 13903-13908 (2007).</li><li>Hayashi, T., Rumbaugh, G., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+AMPA+receptor+subunit+trafficking+by+palmitoylation+of+two+distinct+sites.">Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites.</a> Neuron. 47, 709-723 (2005).</li><li>Lin, D. T., Makino, Y., Sharma, K., Hayashi, T., Neve, R., Takamiya, K., Huganir, R. L. <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+AMPA+receptor+extrasynaptic+insertion+by+4.1N,+phosphorylation.">Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation.</a> 12, 879-887 (2009).</li><li>Araki, Y., Lin, D. T., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+insertion+of+the+AMPA+receptor+GluA2+subunit+is+regulated+by+NSF+binding+and+Q/R+editing+of+the+ion+pore.">Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 11080-11085 (2010).</li><li>Li, Y., Roy, B. D., Wang, W., Zhang, L., Zhang, L. F., Sampson, S. B., Yang, Y. P., Lin, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Two+Functionally+Distinct+Endosomal+Recycling+Pathways+for+Dopamine+D2+Receptor.">Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2 Receptor.</a> Journal of Neuroscience. , (2012).</li><li>Yu, Y. J., Dhavan, R., Chevalier, M. W., Yudowski, G. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+delivery+of+internalized+signaling+receptors+to+the+somatodendritic+surface+by+sequence-specific+local+insertion.">Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion.</a> J. Neurosci. 30, 11703-11714 (2010).</li><li>Yudowski, G. A., Puthenveedu, M. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+modes+of+regulated+receptor+insertion+to+the+somatodendritic+plasma+membrane.">Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane.</a> Nat. Neurosci. 9, 622-627 (2006).</li><li>Yudowski, G. A., Puthenveedu, M. A., Leonoudakis, D., Panicker, S., Thorn, K. S., Beattie, E. C., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+discrete+exocytic+events+mediating+surface+delivery+of+AMPA+receptors.">Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors.</a> J. Neurosci. 27, 11112-11121 (2007).</li><li>Ashby, M. C., De La Rue, S. A., Ralph, G. S., Uney, J., Collingridge, G. L., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+AMPA+receptors+(AMPARs)+from+synapses+is+preceded+by+transient+endocytosis+of+extrasynaptic+AMPARs.">Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs.</a> J. Neurosci. 24 (AMPARs), 5172-5176 (2004).</li><li>Kopec, C. D., Real, E., Kessels, H. W., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GluR1+links+structural+and+functional+plasticity+at+excitatory+synapses.">GluR1 links structural and functional plasticity at excitatory synapses.</a> J. Neurosci. 27, 13706-13718 (2007).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+incorporation+into+synapses+during+LTP:+the+role+of+lateral+movement+and+exocytosis.">AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis.</a> Neuron. 64, 381-390 (2009).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+versus+global+synaptic+plasticity+on+dendrites+controlled+by+experience.">Compartmentalized versus global synaptic plasticity on dendrites controlled by experience.</a> Neuron. 72, 1001-1011 (2011).</li><li>Wang, Z., Edwards, J. G., Riley, N., Provance, D. W., Karcher, R., Li, X. D., Davison, I. G., Ikebe, M., Mercer, J. A., Kauer, J. A., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Vb+mobilizes+recycling+endosomes+and+AMPA+receptors+for+postsynaptic+plasticity.">Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.</a> Cell. 135, 535-548 (2008).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li><li>Sekine-Aizawa, Y., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+receptor+trafficking+by+using+alpha-bungarotoxin-binding-site-tagged+receptors.">Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17114-17119 (2004).</li></ol>

没有利益冲突的声明。

不知什么原因，小鼠神经元总是很难培养的大鼠神经元。在我们的经验中，混合培养的神经元和神经胶质细胞的原代培养的小鼠神经元。然而，这样的混合培养TIRF成像实验是不适合的，在这种类型的培养的神经元和其流程往往顶部的神经胶质细胞上生长的情况，神经元的胞体和树突状进程鞭长莫及TIRF显微镜。因此，较低的密度盖玻片上少数神经胶质细胞的神经元培养TIRF成像的理想选择。我们首?...

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imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons

质膜（PM）和细胞内受体之间贩运的机制更好地理解需要具有出色的空间分辨率和时间分辨率的实验方法。此外，这种方法还必须有分辨能力定位于PM在细胞内的受体。最重要的是，检测的受体，在一个单一的囊泡需要优异的检测灵敏度，因为每个囊泡进行只有一小数量的受体。检查受体活动的标准方法，包括表面生物素的生化检测，缺乏必要的空间和时间的决议;和荧光显微镜检查的immunolabeled表面的受体，这需要细胞化学固定，因此缺乏足够的时间分辨率1-6。为了克服这些限制，我们和其他人开发的，采用了一种新STRAtegy，使可视化动态插入的受体出色的空间分辨率和时间分辨率7-17 PM。该方法包括pH敏感的GFP，superecliptic的pHluorin 18的N-末端胞外结构域的受体的标记。 superecliptic pHluorin荧光在中性pH值和非荧光在酸性pH值（pH值&lt;6.0）的独特属性。因此，标记受体的非荧光内酸性细胞内转运囊泡管腔或内体舱室时，它们变得容易地可视化，只有当暴露于细胞外的中性pH值环境上的外表面的PM。因此，我们的战略使我们能够区分PM表面受体，在细胞内囊泡。为了达到足够的空间和时间分辨率，以及学习所需的动态贩卖受体的敏感性，我们采用全内反映离子荧光显微镜（TIRFM），这使我们能够实现光学成像（〜170 nm）的最佳的空间分辨率，时间分辨率的视频率显微镜（30帧/秒），且灵敏度来检测单个GFP的荧光分子。成像pHluorin标记的受体TIRFM下，我们能够直接可视个人到PM培养的神经元受体插入事件。这种成像方法可能被应用于任何膜蛋白的胞外结构域，可以标记superecliptic pHluorin，并且将允许的密钥的详细机制不同的膜蛋白（受体，离子通道，运输等）插入到夹层PM。

Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

通过标记与superecliptic pHluorin膜受体的胞外域的，并通过成像这些融合在培养的小鼠神经元受体，我们可以直接可视化个别水泡插入事件到质膜的受体。这种技术将有助于阐明受体插入到质膜的分子机制。

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

imaging-phluorin-tagged-receptor-insertion-to-plasma-membrane-primary

Research

JoVE Journal

Neuroscience

13.1K 次观看.  The Jackson Laboratory. 通过标记与superecliptic pHluorin膜受体的胞外域的，并通过成像这些融合在培养的小鼠神经元受体，我们可以直接可视化个别水泡插入事件到质膜的受体。这种技术将有助于阐明受体插入到质膜的分子机制。

视频: Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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 ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...