Name: an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins
Uploaded: 2018-06-26T15:00:00
Description: Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

我们感谢 Irmgard 维斯的专家技术援助。这项工作得到了 DFG 的支持, 通过显微镜在纳米范围和分子生理学的大脑 (CNMPB, B1-7, T.D.) 的卓越集群。

没有研究与活动物进行。有关安乐死动物获得细胞培养的实验得到了当地动物保护当局 (Tierschutzkommission Universitätsmedizin) 的批准, T10/30 批准号。这些实验是通过批准的协议进行的。
1. 初级海马细胞培养
<ol><li>制备大鼠海马的离解细胞培养在胚胎天 1916,17。将12毫米盖玻片上的细胞涂上聚乙烯亚胺 (裴), 在24井的菜肴中, 密度为 5万-6?...

<ol>
	<li>Koh, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+I,+a+Ca2++sensor+for+neurotransmitter+release.">Synaptotagmin I, a Ca2+ sensor for neurotransmitter release.</a> Trends in Neurosciences. 26, 413-422 (2003).</li><li>Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Does+Synaptotagmin+Trigger+Neurotransmitter+Release.">How Does Synaptotagmin Trigger Neurotransmitter Release.</a> Annual Review of Biochemistry. 77, 615-641 (2008).</li><li>Perin, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Functional+Conservation+of+Synaptotagmin+(p65)+in+Drosophila+and+Humans.">Structural and Functional Conservation of Synaptotagmin (p65) in Drosophila and Humans.</a> Journal of Biological Chemistry. 266, 615-622 (1991).</li><li>Matteoli, M., Takei, K., Perrin, M. S., S&#252;dhof, T. C., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exo-endocytotic+Recycling+of+Synaptic+Vesicles+in+Developing+Processes+of+Cultured+Hippocampal+Neurons.">Exo-endocytotic Recycling of Synaptic Vesicles in Developing Processes of Cultured Hippocampal Neurons.</a> Journal of Cell Biology. 117, 849-861 (1992).</li><li>Ko, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-1+performs+neurexin-dependent+and+neurexin-independent+functions+in+synapse+validation.">Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation.</a> The EMBO Journal. 28, 3244-3255 (2009).</li><li>Shinoda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+enhances+spontaneous+and+activity-dependent+neurotransmitter+release+at+excitatory+terminals+but+not+at+inhibitory+terminals+in+hippocampal+neurons.">BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.</a> Frontiers in Synaptic Neuroscience. 6, 27 (2014).</li><li>Ivanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+controls+localization+and+function+of+CtBP+1+via+binding+to+Bassoon+and+Piccolo.">Synaptic activity controls localization and function of CtBP 1 via binding to Bassoon and Piccolo.</a> The EMBO Journal. 34, 1056-1077 (2015).</li><li>Crawford, D. C., Ramirez, D. M. O., Trauterman, B., Monteggia, L. M., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+molecular+impairment+of+spontaneous+neurotransmission+modulates+synaptic+efficacy.">Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy.</a> Nature Communications. 8, 1-14 (2017).</li><li>Riemann, D., Wallrafen, R., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kohlsch&#252;tter-T&#246;nz+syndrome+associated+gene+Rogdi+encodes+a+novel+presynaptic+protein.">The Kohlsch&#252;tter-T&#246;nz syndrome associated gene Rogdi encodes a novel presynaptic protein.</a> Scientific Reports. 7, (2017).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rogdi+Defines+GABAergic+Control+of+a+Wake-promoting+Dopaminergic+Pathway+to+Sustain+Sleep+in+Drosophila.">Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila.</a> Scientific Reports. 7, 1-14 (2017).</li><li>Schossig, A., Wolf, N. I., Kapferer, I., Kohlsch&#252;tter, A., Zschocke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic+encephalopathy+and+amelogenesis+imperfecta:+Kohlsch&#252;tter-T&#246;nz+syndrome.">Epileptic encephalopathy and amelogenesis imperfecta: Kohlsch&#252;tter-T&#246;nz syndrome.</a> Euopean Journal of Medical Genetics. 55, 319-322 (2012).</li><li>Kraszewski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicle+Dynamics+in+Living+Cultured+Hippocampal+Neurons+Visualized+with+CY3-Conjugated+Antibodies+Directed+against+the+Lumenal+Domain+of+Synaptotagmin.">Synaptic Vesicle Dynamics in Living Cultured Hippocampal Neurons Visualized with CY3-Conjugated Antibodies Directed against the Lumenal Domain of Synaptotagmin.</a> Journal of Neuroscience. 15, 4328-4342 (1995).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Petkova, A., Goedecke, N., Korte, M., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligins+mediate+presynaptic+maturation+through+brain-derived+neurotrophic+factor+signaling.">Neuroligins mediate presynaptic maturation through brain-derived neurotrophic factor signaling.</a> bioRxiv. , 262246 (2018).</li><li>Fuchs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(A)+receptors+can+initiate+the+formation+of+functional+inhibitory+GABAergic+synapses.">GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses.</a> European Journal of Neuroscience. 38, 3146-3158 (2013).</li><li>Dresbach, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+regions+of+the+presynaptic+cytomatrix+protein+Bassoon:+Significance+for+synaptic+targeting+and+cytomatrix+anchoring.">Functional regions of the presynaptic cytomatrix protein Bassoon: Significance for synaptic targeting and cytomatrix anchoring.</a> Molecular and Cellular Neuroscience. 23, 279-291 (2003).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Hippocampal+Neurons+from+Prenatal+Mice.">Isolation and Culture of Hippocampal Neurons from Prenatal Mice.</a> Journal of Visualized Experiments. (65), 4-9 (2012).</li><li>Tsuriel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+sharing+as+a+predominant+determinant+of+synaptic+matrix+molecular+dynamics.">Local sharing as a predominant determinant of synaptic matrix molecular dynamics.</a> PLOS Biology. 4, 1572-1587 (2006).</li><li>Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Synaptic+Vesicle+Recycling+Using+FM+Dyes+During+Evoked,+Spontaneous,+and+Miniature+Synaptic+Activities.">Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities.</a> Journal of Visualized Experiments. (85), 1-10 (2014).</li><li>Villarreal, S., Lee, S. H., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Synaptic+Vesicle+Endocytosis+in+Cultured+Hippocampal+Neurons.">Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.</a> Journal of Visualized Experiments. (127), 1-8 (2017).</li><li>Kavalali, E. T., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+presynaptic+function.">Visualizing presynaptic function.</a> Nature Neuroscience. 17, 10-16 (2014).</li><li>Opazo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+Intermixing+of+Synaptic+Vesicle+Components+upon+Vesicle+Recycling.">Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling.</a> Traffic. 11, 800-812 (2010).</li><li>Wollebo, H. S., Woldemichaele, B., White, 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=Lentiviral+transduction+of+neuronal+cells.">Lentiviral transduction of neuronal cells.</a> Methods in Molecular Biology. 1078, 141-146 (2013).</li><li>Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexin+Clamps+Asynchronous+Release+by+Blocking+a+Secondary+Ca2++Sensor+via+Its+Accessory+&#945;+Helix.">Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory &#945; Helix.</a> Neuron. 68, 907-920 (2010).</li><li>Wittenmayer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+Neuroligin1+regulates+presynaptic+maturation.">Postsynaptic Neuroligin1 regulates presynaptic maturation.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13564-13569 (2009).</li><li>Lazarevic, V., Schone, C., Heine, M., Gundelfinger, E. D., Fejtova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Remodeling+of+the+Presynaptic+Cytomatrix+upon+Homeostatic+Adaptation+to+Network+Activity+Silencing.">Extensive Remodeling of the Presynaptic Cytomatrix upon Homeostatic Adaptation to Network Activity Silencing.</a> Journal of Neuroscience. 31, 10189-10200 (2011).</li><li>Kraszewski, K., Daniell, L., Mundigl, O., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+synaptic+vesicles+in+nerve+endings+monitored+by+recovery+from+photobleaching+of+synaptic+vesicle-associated+fluorescence.">Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.</a> Journal of Neuroscience. 16, 5905-5913 (1996).</li><li>Hua, Y., Sinha, R., Martineau, M., Kahms, M., Klingauf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+origin+of+synaptic+vesicles+undergoing+evoked+and+spontaneous+fusion.">A common origin of synaptic vesicles undergoing evoked and spontaneous fusion.</a> Nature Neuroscience. 13, 1451-1453 (2010).</li><li>Han, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycosylation+Is+Essential+for+Vesicular+Targeting+of+Synaptotagmin+1.">N-Glycosylation Is Essential for Vesicular Targeting of Synaptotagmin 1.</a> Neuron. 41, 85-99 (2004).</li><li>Kwon, S. E., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+is+dispensable+for+sorting+of+synaptotagmin+1+but+is+critical+for+targeting+of+SV2+and+synaptophysin+to+recycling+synaptic+vesicles.">Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles.</a> Journal of Biological Chemistry. 287, 35658-35668 (2012).</li><li>Afuwape, O. A. T., Wasser, C. R., Schikorski, T., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pool-specific+modification+of+neurotransmitter+release+by+intravesicular+free+radical+generation.">Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.</a> The Journal of Physiology. 595, 1223-1238 (2017).</li><li>Sara, Y., Virmani, T., De&#225;k, F., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+pool+of+vesicles+recycles+at+rest+and+drives+spontaneous+neurotransmission.">An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission.</a> Neuron. 45, 563-573 (2005).</li><li>Wilhelm, B. G., Groemer, T. W., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+synaptic+vesicles+drive+active+and+spontaneous+release.">The same synaptic vesicles drive active and spontaneous release.</a> Nature Neuroscience. 13, 1454-1456 (2010).</li><li>Bacci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+blockade+of+glutamate+receptors+enhances+presynaptic+release+and+downregulates+the+interaction+between+synaptophysin-synaptobrevin-vesicle-associated+membrane+protein+2.">Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.</a> Journal of Neuroscience. 21, 6588-6596 (2001).</li><li>Piccoli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+Controls+Synaptic+Vesicle+Storage+and+Mobilization+within+the+Recycling+Pool.">LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool.</a> Journal of Neuroscience. 31, 2225-2237 (2011).</li><li>Tracy, T. E., Yan, J. J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+knockdown+of+AMPA+receptors+reveals+a+trans-synaptic+signal+for+presynaptic+maturation.">Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation.</a> The EMBO Journal. 30, 1577-1592 (2011).</li><li>Truckenbrdot, S., Viplav, A., Jaehne, S., Vogts, A., Denker, A., Wildhagen, H., Fornasiero, E. F., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+synaptic+vesicles+are+inactivated+by+contamination+with+SNAP25.">Ageing synaptic vesicles are inactivated by contamination with SNAP25.</a> bioRxiv. , 172239 (2017).</li></ol>

有三项常规用于研究突触囊泡 (SV) 回收。前两项包括使用 a) 荧光苯乙烯染料, 如 FM1-43 (纳入膜, 在吞期间被纳入细胞器, 并在胞吐作用后释放);和 b) 荧光标记重组 SV 蛋白 (在过度表达后, 纳入蛋白质回收机械)。如果附加的显影根据 pH 值改变荧光, 它们可以用来监测 SV 酸性内部和胞外培养基 ph 值的变化。用 GFP 的 pH 敏感变种标记的重组蛋白称为 Phluorins。所讨论的两种分析方法都有以前...

研究突触囊泡回收在确定突触前的性质如何改变, 无论是在突触可塑性或响应突触功能的扰动。研究 Synaptotagmin-1 (Syt1) 抗体摄取提供了一种测量 SV 循环量的方法。Syt1 是一种 SV 相关蛋白, 充当 Ca2 +传感器, 是 exocytotic 释放神经递质1,2的必要条件。它是一种跨膜蛋白, 具有 C 末端细胞质域外的 sv 和 n-端腔内域的 sv3。在胞吐作用过程中, Syt1 的腔域会暴露于外界介质中。对于这个外在的媒介, 我们增加抗体针对细胞质领域, 在吞期间被内部化。这些抗体可以是预共轭与显影或 immunostained 与二级抗体4,5,6,7。结果 immunosignal 的荧光强度与 SV 回收量成正比。这种方法可用于确定本构和退极化诱发 SV 循环6,8。
Syt1 摄取化验可以执行后, 病毒介导的基因转移到几乎所有的细胞在盘子里或经过稀疏转染少量的细胞。本方法将原发海马神经元的稀疏双转染法与磷酸钙9结合使用。我们使用一种已知的重组标记蛋白积累在 presynapses, 荧光标记突触素, 找到突触前终端和过度表达 tshr 我们的蛋白质的兴趣, Rogdi。这使我们可以测试是否 Rogdi 目标功能突触和影响 SV 回收。基因编码 Rogdi 最初是在屏幕上发现的果蝇突变体的特点是受损的记忆10。在人类中, Rogdi 基因突变导致一种罕见的破坏性疾病, 称为 Kohlschütter-Tönz 综合征。患者患有牙釉质畸形、pharmacoresistant 癫痫、精神运动迟缓;然而, 基因产物的亚细胞定位仍然是难以捉摸的11。因此, Syt1 摄取检测为 GFP 标记的 Rogdi 在功能突触9的定位提供了关键的证据。
这种吸收技术有几个好处。首先, 通过测量 Syt1 荧光标签的荧光强度, 可以实时观测到7、12、6、9的活度成像, 从而实现 SV 循环。此外, 还开发了几种 Syt1 抗体变种。有未加标签的变体, 可以在固定后使用标准染色协议标记为继发抗体, 并附有荧光标签的预共轭变种。最后, 抗体为基础的荧光是有利的, 因为大量选择的商业可用的二级或共轭染料, 可以使用。
当固定和染色神经元时, 也有可能染色额外的蛋白质和进行定位分析。这可以帮助确定它们的位置与回收 SVs 的关系。荧光标签的强度是 SV 回收量的直接度量。此外, 抗体有选择地标记 Syt1-containing 结构, 导致高特异性和小背景荧光4。也可以使用不同的刺激协议, 如退极化缓冲器或电刺激协议9,12,13,14。但是, 在不刺激神经元培养15的情况下, 可以测量基底 SV 循环。
我们的方法专门解决了双转染神经元的 Syt1 抗体摄取与继发抗体 immunolabeling 固定后。但是, 我们在讨论中提到了所有常规使用的方法变体, 使观众有机会根据具体需要调整该协议。

<table>
	<tbody>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Invitrogen</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Merck</td>
			<td>8.03456.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti mouse Alexa 647</td>
			<td>Jackson-Immunoresearch</td>
			<td>715605151</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td>087721H</td>
			<td></td>
		</tr>
		<tr>
			<td>tube (50 mL)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>BiochromAG</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>1,083,421,000</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Hera Cell 150 (Inkubator)</td>
			<td colspan="2">ThermoElectron Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Honeywell</td>
			<td>M0250-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Fisher Scientific</td>
			<td>10144633CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioFroxx</td>
			<td>1394KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OpenView Experiment Analysis Application</td>
			<td>Free software, see comments</td>
			<td></td>
			<td>written by Noam E. Ziv, Technion &#8211; Israel Institute of Technology, Haifa, Israel</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Penstrep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P6148-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>safety hood</td>
			<td>ThermoElectron</td>
			<td></td>
			<td>Serial No. 40649111</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>neoFroxx</td>
			<td>1104kg001</td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptotagmin1</td>
			<td>Synaptic Systems</td>
			<td>105311</td>
			<td>mouse monoclonal; clone 604.2</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1,086,031,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Observer. Z1&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

在主动突触前神经终端, 突触囊泡经历了外周和吞的循环。在回收过程中, SV 跨膜蛋白的腔域在细胞表面暴露。其中一种蛋白质是 Synaptotagmin-1 (Syt1)。对 Syt1 的腔域进行的抗体, 一旦添加到培养基中, 就会在 endocytotic 周期内被占用。这种吸收量与 SV 循环的数量成正比, 可以通过免疫荧光进行量化。在这里, 我们结合 Syt1 抗体摄取与双转染培养海马神经元。这使我们能够 (1) 根据重组突触前标记突触素的表达来本地化突触前站点, (2) 使用 Syt1 摄取确定它们的功能, (3) 描述了一种感兴趣的蛋白质的靶向性和作用, GFP-Rogdi。

这种方法的预期结果是每盖玻片大约有50个双转染神经元, 每一个密度为5万个神经元。每个神经元的轴突将显示荧光标记的突触素堆积的多个热点, 表明簇 ofSVs。在功能突触前部位, 重组突触素信号 colocalizes 有点 Syt1 荧光。使用双转染, 无论是 GFP-Rogdi 作为感兴趣的蛋白质 (图 1) 或 mGFP, 因为该控制与重组突触素共同表达。
<p class="jove_content" fo:keep-tog...

Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

我们描述了一种用于培养神经元的突触囊泡 (SV) 循环的光学检测方法。将该协议与双转染相结合, 以表达突触前标记和感兴趣的蛋白质, 使我们能够定位突触前的部位, 它们的突触囊泡再生能力, 并确定感兴趣的蛋白质的作用。

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Research

JoVE Journal

Neuroscience

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

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

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

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

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

使用调频染料诱发过程中，自发的，微型突触活动检查突触囊泡循环的

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...