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Neurosciences

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

Published: June 26, 2018
doi:
10.3791/58043
, , ,
1Institute for Anatomy and Embryology,University Medical Centre Göttingen

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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?…

Representative Results

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…

Discussion

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Optical AssaySynaptic Vesicle RecyclingCultured NeuronsPresynaptic ProteinsAntibody SpecificityPrimary Hippocampal CellsTransfectionCalcium ChlorideEndotoxin-free DNATransfection BufferReduced-serum MediumCell-culture MediumIncubationWashingActive Synapses
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Riemann, D., Petkova, A., Dresbach, T., Wallrafen, R. An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins. J. Vis. Exp. (136), e58043, doi:10.3791/58043 (2018).
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