海马神经元中突触囊泡吞的测量
Summary
突触囊泡吞是通过光学显微镜检测 pHluorin 融合与突触泡蛋白和电子显微镜的囊泡摄取。
Abstract
在吞, 融合突触泡在神经末端被检索, 允许囊泡再循环和因而维持突触传输在重复的神经生火期间。损伤的吞在病理条件下导致突触强度和脑功能下降。在这里, 我们描述的方法用于测量突触囊泡吞在哺乳动物海马突触在神经元培养。我们监测突触囊泡蛋白吞融合的突触囊膜蛋白, 包括突和 VAMP2/synaptobrevin, 在水泡腔侧, 与 pHluorin, pH 敏感的绿色荧光蛋白, 增加其荧光强度随着 pH 值的增加而增大。在胞, 水泡流明 ph 增加, 而在吞水泡流明 ph 值是 re-acidified。因此, pHluorin 荧光强度的增加表明融合, 而减少表明吞的标签突触泡蛋白。除了使用 pHluorin 成像方法记录吞, 我们还通过电子显微镜 (EM) 测量了辣根过氧化物酶 (HRP) 对囊泡的摄取量来监测水泡膜的吞。最后, 在高钾诱导去极化后的不同时间, 我们监测了神经末端膜凹坑的形成。HRP 吸收和膜坑形成的时间过程表明吞的时间过程。
Introduction
神经递质储存在突触泡中, 由胞释放。突触囊泡膜和蛋白质被吞化, 并在下一轮胞中重复使用。突触囊泡的吞对维持突触囊泡池和从细胞膜上去除突起的泡囊很重要。ph 敏感的绿色荧光蛋白 pHluorin, 这是在酸性情况下淬火和 dequenched 在中性 ph 值, 已被用来测量吞时间课程在活细胞1,2,3。pHluorin 蛋白通常附着在突触囊泡蛋白的腔侧, 如突或 VAMP2/synaptobrevin。在休息, pHluorin 是淬火在 5.5 pH 流明的突触泡。囊泡融合到等离子体膜暴露水泡腔的胞外溶液的 pH 值是〜 7.3, 导致增加 pHluorin 荧光。胞后, 增加的荧光衰变, 由于吞的突触囊泡蛋白, 其次是囊泡 re-acidification 在这些回收泡。虽然衰变反映了吞和水泡 re-acidification, 它主要反映吞, 因为 re-acidification 在大多数情况下比吞快1,4。re-acidification 的时间常数为 3-4 s 或更少的5,6, 它通常比十年代或更多的泡吞4,5所需的速度快。如果需要进行实验来区分吞和 re-acidification, 使用 4-Morpholineethanesulfonic 酸 (MES) 溶液 (25 mM) 和 pH 值为5.5 的酸性淬火实验可以用来确定是否检索突触囊泡蛋白从等离子膜通过吞1,3,4。因此, pHluorin 荧光强度的增加反映了外、吞的平衡, 神经刺激后的减少具体反映了吞。
pHluorin 成像不仅可以用来测量吞的时间过程, 也可用于突触囊泡池的大小7,8, 以及诱发释放和自发释放9的概率。许多因素和蛋白质参与调节吞, 如钙, 可溶性 NSF-附着蛋白受体 (诱捕) 蛋白, 脑源性神经营养因子 (BDNF) 和磷酸已被确定使用 pHluorin 成像1,2,10,11,12,13,14,15,16. 此外, 神经递质的释放不仅可在原发神经元中检测, 而且可以在显微镜17的神经母细胞瘤中发现。最近, pHluorin 变体、dsRed、mOrange 和 pHTomato 被开发用于在单个突触1819中监视多个因素的同时录制。例如, pHTomato 已与突融合, 并与基因编码的钙指示剂 (GCaMP5K) 一起使用, 以监测前囊泡融合, 并在突触后室20中的 Ca2 +流入。因此, pHluorin 附在突触蛋白提供了一个有用的方法来分析吞和胞的关系。
EM 是另一种方法, 通常用于研究吞, 由于高空间分辨率, 显示超微结构的变化, 在吞。两个一般的领域是能够可视化的病理改变神经元细胞21和跟踪囊泡蛋白22。特别是, 观察突触囊泡摄取, 膜曲率涂覆格在 periactive 区, 和内涵结构是可能的 EM3,23,24,25 ,26,27,28。虽然 EM 涉及潜在的文物, 如固定诱发的畸形, 可能会影响吞, 和数据分析是劳动密集型, 该决议提供了一个诱人的机会, 可视化细胞结构。在吞27期间, 可能存在的定影问题和 EM 时间分辨率的限制可以通过高压冻结来克服, 提供一种快速和非化学的方法来稳定当前的微妙结构。
Protocol
注意: 下面的协议描述了在培养海马神经元中使用的 pHluorin 成像方法和 em 方法. pHluorin 监测活细胞中突触囊泡蛋白的摄取和 em 检测突触泡的摄取和超微结构的变化. 动物护理和程序遵循 nih 的指导方针, 并得到了 nih 动物保育和使用委员会的批准. 1. pHluorin 成像 海马神经元培养 通过组合 4 mm NaHCO 3 和 5 mm HEPES, 并?…
Representative Results
使用脂质载体法, SpH 表达了海马神经元, 允许识别 boutons (图 1a)。电刺激细胞诱导胞, 并相应增加荧光强度。荧光 (δ) 的增加是通过终止刺激 (图 1b) 停止的。随着吞的增加, 荧光量也随之缓慢下降。在 VAMP2-pHluorin 的情况下, VAMP2 扩散沿轴突从一个溥后刺激4。原始数据使用任意单位, 并被规范化到基线以获得衰变?…
Discussion
这里我们展示了两种监测突触囊泡吞的方法。在第一种方法中, 我们监测 pHluorin 融合的突触泡蛋白在转染神经元和随后电刺激。其次, 我们使用的 EM 成像的 HRP 吸收诱导氯化钾。我们使用不同的刺激有两个原因。首先, 高钾的应用诱导了培养中所有神经元的去极化。这有助于 em 检查, 因为我们的 em 方法不能区分陶土和受激神经元。其次, 微的形态学变化更可靠地观察后, 强烈的刺激, 如高钾刺激, 而…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 Dr. 的 synaptophysin-pHluorin2x 建造, Dr. 提供 VAMP2-phluorin。我们感谢 Dr. 苏珊和弗吉尼亚克罗克的一电子显微镜设施的技术支持和帮助。这项工作得到了美国国家神经疾病研究所和脑卒中内部研究计划的支持, 并得到了 KRIBB 研究计划 (韩国生物医学科学家研究项目) 的资助, 韩国研究所大韩民国生物科学和生物工程。
Materials
| Lipofectamine LTX with Plus | Thermo Fisher | 15338-100 | Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin |
| neurobasal medium | Thermo Fisher | 21103-049 | Growth medium for neuron, Warm up to 37°C before use |
| B27 | Thermo Fisher | 17504-044 | Gradient for neuronal differentiation |
| Glutamax | Thermo Fisher | 35050-061 | Gradient for neuronal culture |
| Poly-D-Lysine coated coverslip | Neuvitro | GG-25-pdl | Substrate for neuronal growth and imaging of pHluorin |
| Trypsin XI from bovine pancrease | Sigma | T1005 | Neuronal culture-digest hippocampal tissues |
| Deoxyribonuclease I from bovine pancreas | Sigma | D5025 | Neuronal culture-inhibits viscous cell suspension |
| pulse stimulator | A-M systems | model 2100 | Apply electrical stimulation |
| Slotted bath with field stimulation | Warner Instruments | RC-21BRFS | Apply electrical stimulation |
| stimulus isolation unit | Warner Instruments | SIU102 | Apply electrical stimulation |
| lubricant | Dow corning | 111 | pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber |
| AP5 | Tocris | 3693 | Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity |
| CNQX | Tocris | 190 | Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity |
| Illuminator | Nikon | C-HGFI | Metal halide light source for pHluorin |
| EMCCD camera | Andor | iXon3 | pHluorin imaging, detect pHluorin fluorescence intensity |
| Inverted microscopy | Nikon | Ti-E | Imaging for synaptophysin or VAMP2 pHluorin transfected cells |
| NIS-Elements AR | Nikon | NIS-Elements Advanced Research | Software for imaging acquisition and analysis |
| Igor Pro | WaveMetrics | Igor pro | Software for imaging analysis and data presentation |
| imaging chamber | Warner Instruments | RC21B | pHluorin imaging, apply field stimulation on living cells |
| poly-l-lysine | Sigma | P4832 | Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times |
| Horseradish peroxidase(HRP) | Sigma | P6782 | Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use |
| Na cacodylate | Electron Microscopy Sciences | 12300 | Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N |
| 3,3′-Diaminobenzidine(DAB) | Sigma | D8001 | Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use |
| Hydrogen peroxide solution | Sigma | H1009 | Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use |
| glutaraldehyde | Electron Microscopy Sciences | 16365 | Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use |
| TEM | JEOL | 200CX | Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes |
| CCD digital camera | AMT | XR-100 | Electron microscopy, capturing images |
| Lead citrate | Leica microsystems | 16707235 | Electron microscopy, grid staining |
Referências
- Sankaranarayanan, S., Ryan, T. A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nature cell biol. 2 (4), 197-204 (2000).
- Sun, T., Wu, X. S., et al. The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses. J Neurosci. 30 (35), 11838-11847 (2010).
- Wu, X. -. S. S., Lee, S. H., et al. Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses. Neuron. 92 (5), 1020-1035 (2016).
- Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron. 51 (6), 773-786 (2006).
- Atluri, P. P., Ryan, T. A. The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals. J Neurosci. 26 (8), 2313-2320 (2006).
- Royle, S. J., Granseth, B., Odermatt, B., Derevier, A., Lagnado, L. Imaging phluorin-based probes at hippocampal synapses. Methods Mol Biol. 457, 293-303 (2008).
- Moulder, K. L., Mennerick, S. Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons. J Neurosci. 25 (15), 3842-3850 (2005).
- Li, Z., Burrone, J., Tyler, W. J., Hartman, K. N., Albeanu, D. F., Murthy, V. N. Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc Natl Acad Sci U S A. 102 (17), 6131-6136 (2005).
- Morris, R. G. Elements of a neurobiological theory of hippocampal function: the role of synaptic plasticity, synaptic tagging and schemas. Eur J Neurosci. 23 (11), 2829-2846 (2006).
- Sankaranarayanan, S., Ryan, T. A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat Neurosci. 4 (2), 129-136 (2001).
- Balaji, J., Armbruster, M., Ryan, T. A. Calcium control of endocytic capacity at a CNS synapse. J Neurosci. 28 (26), 6742-6749 (2008).
- Ferguson, S. M., Brasnjo, G., et al. A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science. 316 (5824), 570-574 (2007).
- Baydyuk, M., Wu, X. S., He, L., Wu, L. G. Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal. J Neurosci. 35 (11), 4676-4682 (2015).
- Wu, X. S., Zhang, Z., Zhao, W. D., Wang, D., Luo, F., Wu, L. G. Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells. Cell Rep. 7 (4), 982-988 (2014).
- Zhang, Z., Wang, D., et al. The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses. J Neurosci. 33 (21), 9169-9175 (2013).
- Wu, L. -. G. G., Hamid, E., Shin, W., Chiang, H. -. C. C. Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu Rev Physiol. 76 (1), 301-331 (2014).
- Daniele, F., Di Cairano, E. S., Moretti, S., Piccoli, G., Perego, C. TIRFM and pH-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: cell imaging and data analysis. J Vis Exp. (95), e52267 (2015).
- Shaner, N. C., Lin, M. Z., et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods. 5 (6), 545-551 (2008).
- Li, Y., Tsien, R. W. pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci. 15 (7), 1047-1053 (2012).
- Leitz, J., Kavalali, E. T. Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. Elife. 3, e03658 (2014).
- Bisht, K., El Hajj, H., Savage, J. C., Sánchez, M. G., Tremblay, M. -. &. #. 2. 0. 0. ;. Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques. J Vis Exp. (112), e54060 (2016).
- Schikorski, T. Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope. High-Resolution Imaging of Cellular Proteins: Methods and Protocols. , 327-341 (2016).
- Kononenko, N. L., Puchkov, D., et al. Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron. 82 (5), 981-988 (2014).
- Wu, Y., O’Toole, E. T., et al. A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis. eLife. 2014 (3), e01621 (2014).
- Heuser, J. E., Reese, T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J Cell Biol. 57 (2), 315-344 (1973).
- Ceccarelli, B., Hurlbut, W. P., Mauro, A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J Cell Biol. 57 (2), 499-524 (1973).
- Watanabe, S., Rost, B. R., et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature. 504 (7479), 242-247 (2013).
- Watanabe, S., Trimbuch, T., et al. Clathrin regenerates synaptic vesicles from endosomes. Nature. 515 (7526), 228-233 (2014).
- Sankaranarayanan, S., Atluri, P. P., Ryan, T. A. Actin has a molecular scaffolding, not propulsive, role in presynaptic function. Nat Neurosci. 6 (2), 127-135 (2003).
- Zhu, Y., Xu, J., Heinemann, S. F. Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging. Neuron. 61 (3), 397-411 (2009).
- Miesenbock, G., De Angelis, D. A., Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 394 (6689), 192-195 (1998).
- Arnao, M. B. B., Acosta, M., del Rio, J. A. A., García-Cánovas, F. Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent. Biochim Biophys Acta. 1038 (1), 85-89 (1990).
- Deák, F., Schoch, S., et al. Synaptobrevin is essential for fast synaptic-vesicle endocytosis. Nat Cell Biol. 6 (11), 1102-1108 (2004).
- Clayton, E. L., Evans, G. J. O., Cousin, M. A. Bulk synaptic vesicle endocytosis is rapidly triggered during strong stimulation. J Neurosci. 28 (26), 6627-6632 (2008).
- Kavalali, E. T., Jorgensen, E. M. Visualizing presynaptic function. Nat Neurosci. 17 (1), 10-16 (2014).
- Wienisch, M., Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat Neurosci. 9 (8), 1019-1027 (2006).
- Fernández-Alfonso, T., Kwan, R., Ryan, T. A. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron. 51 (2), 179-186 (2006).
- Gimber, N., Tadeus, G., Maritzen, T., Schmoranzer, J., Haucke, V. Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins. Nat Commun. 6, 8392 (2015).
- Nicholson-Fish, J. C., Smillie, K. J., Cousin, M. A. Monitoring activity-dependent bulk endocytosis with the genetically-encoded reporter VAMP4-pHluorin. J Neurosci Methods. 266, 1-10 (2016).
- Burrone, J., Li, Z., Murthy, V. N. Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin. Nat Protoc. 1 (6), 2970-2978 (2006).
- Wisse, E., Braet, F., et al. Fixation methods for electron microscopy of human and other liver. World journal of gastroenterology. 16 (23), 2851-2866 (2010).
- Magidson, V., Khodjakov, A. Circumventing Photodamage in Live-Cell Microscopy. Methods in Cell Biology. 114, 545-560 (2013).
- Søndergaard, C. R., Garrett, A. E., et al. Structural artifacts in protein-ligand X-ray structures: implications for the development of docking scoring functions. J Med Chem. 52 (18), 5673-5684 (2009).
Citar este artigo
Villarreal, S., Lee, S. H., Wu, L. Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (127), e55862, doi:10.3791/55862 (2017).