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Oct 2019
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Investigate Synaptic Vesicles Mobility in Neuronal Culture Axons by FRAP Imaging
用FRAP成像技术研究神经元轴突中突触囊泡的移动   

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Abstract

Synaptic vesicles (SVs) are clustered in the presynaptic terminals and consistently trafficking along axons. Based on their release features, SVs are classified into different “pools”. Imaging of SVs that are traveling among multiple presynaptic terminals has helped define a new pool named “SV super-pool”. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) approach to elucidate the relationship between SVs from the super-pool with SV clusters at presynaptic terminals. This method is powerful to investigate SV mobility regulation mechanisms.

Keywords: Synaptic vesicle (突触泡), Membrane trafficking (膜运输), Live-cell imaging (活细胞成像), FRAP (光漂白后的荧光恢复技术), Axonal transport (轴突运输), Synapse (突触)

Background

Synaptic vesicles (SVs) are key organelles involved in neurotransmission through the storage and release of neurotransmitters. SVs are mostly identified in a cluster adjacent to the active zone of the presynaptic terminals. SVs have a homogeneous appearance with a diameter of 40-50 nm under electron microscopy (EM) (Landis et al., 1988; Korogod et al., 2015). While, to our knowledge, there is no significant biochemical distinction between SVs. Under different stimulation paradigms, they show different release properties. Thus, SVs were classified into different functional pools: the reserve pool, the recycling pool and the readily releasable pool (Figure 1) (Denker and Rozzoli, 2010). The detailed synapse structure, SV localization, SV release mechanisms were intensively studied with EM. SVs were found linked to one or two neighboring vesicles by filaments, and synapsins were thought to be part of the connector and maintain the SVs in the reserve pool (Siksou et al., 2007). The dissection of molecular steps for SVs docking and fusion was also revealed by ultrastructure study (Imig et al., 2014). The fusion of SV with the plasma membrane exposes the acidic lumen (pH around 5.0) to the neutral extracellular medium (pH of 7.4) (Südhof, 1995). Therefore, SVs recycling and related molecular mechanisms were also intensively studied by labeling SVs with pH sensors (Sankaranarayanan et al., 2000; Soykan et al., 2017).



Figure 1. Synaptic vesicle pools. The classical model of synaptic vesicle (SV) pools are the reserve pool, the recycling pool, and the readily releasable pool. A newly defined “super-pool” comprises SVs that share among en-passant presynaptic boutons along the axon.


For many years, it was thought that SVs recycle inside a single presynaptic terminal without major exchange with neighboring en-passant boutons of the same axon, while terminals may function with significant autonomy from the distant cell soma. In the past decade, SVs were observed trafficking and sharing between multiple en-passant presynaptic boutons along the axon. This axonal SV population was designated as “super-pool” (Figure 1) (Denker and Rozzoli, 2010). This super-pool was observed through both in vitro and in vivo paradigms (Staras et al., 2010; Herzog et al., 2011; Zhang et al., 2019). Yet, how SVs in super-pool contribute to the neurotransmission remains largely unknown. We could recently show that, changes in the super-pool size impact spontaneous release frequency (Zhang et al., 2019).


FRAP is normally used for determining the kinetics of cell membrane diffusion or protein binding (Axelrod et al., 1976; Qin et al., 2008), while the kinetics of SV mobility is unlike these cases. However, as described above, a certain number of SVs are clustered in each presynaptic bouton and consistently refresh with SVs in super-pool. FRAP allows to measure diffusion of SVs between the presynaptic bouton and the axon. Here, we detail a protocol for FRAP imaging that is able to elucidate the exchange of SVs between the super-pool and presynaptic SV clusters. SVs are labeled with a synaptic vesicle protein tagged with a fluorescent protein, such as VGLUT1venus or Synaptobrevin 2EGFP (Figures 2A-2D). This FRAP method can be applied to both in vitro and in vivo systems. In this paper, we will describe the detailed protocol applied to dissociated hippocampal neuronal culture. This FRAP imaging method will be useful for further explorations of SV mobility related mechanisms.



Figure 2. FRAP sequences acquisition and analysis. In neurons, SVs are labeled by fluorescent protein Venus/EGFP. A presynaptic bouton contains a cluster of SVs, this is shown as a bright individual bouton. A. The experiment work flow. The virus transduction is performed on DIV 1/2 of the culture, and the FRAP imaging is acquired from DIV 17 to 21. Detailed FRAP imaging procedure is showed in the yellow box. B. Randomly select a field of culture for imaging. In the field, select 5 boutons (labeled as red circles) that are distant from each other as ROIs for bleaching. C. The frame acquired just after photobleaching. The fluorescent intensity of 5 selected boutons is significantly decreased due to the photobleaching. D. A frame acquired 1 h after photobleaching, the ROIs fluorescent intensity is gradually recovered. E. Analyze the FRAP sequence with FRAP Analysis Macro. Following menus on the pop-up windows, select the regions of bleached boutons (red circles), the cells in the field for photobleaching correction (yellow region), and the background area for background subtraction (blue region).


Materials and Reagents

  1. Round cover glass, #1 thickness, 18 mm (Warner Instruments, catalog number: 64-0384 )

  2. 35 mm Glass bottom dish with 10 mm micro-well (Cellvis, catalog number: D35-10-1-N )

  3. 15 ml Conical centrifuge tubes (FalconTM, catalog number: 14-959-53A )

  4. 200 µl pipette tips (QuickRack, catalog number: NC9640144 )

  5. C57/BL6J mice (The Jackson Laboratory, catalog number: 000664 )

  6. Leibovitz’s L-15 medium (Gibco, catalog number: 11415064 ), store at 4 °C

  7. 0.05% trypsin-EDTA (Gibco, catalog number: 25300054 ), store at -20 °C

  8. Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, catalog number: 61965026 ), store at 4 °C

  9. Fetal bovine serum (Eurobio, catalog number: CVFSVF001 ), store at -20 °C

  10. Penicillin-streptomycin (Gibco, catalog number: 15140122 ), store at -20 °C

  11. Poly-L-lysine (Sigma, catalog number: P2636 ), store at -20 °C

  12. Neurobasal A medium (Gibco, catalog number: 12349105 ), store at 4 °C

  13. B27 supplement (Gibco, catalog number: 17504044 ), store at -20 °C

  14. Glutamax (Gibco, catalog number: 35050038 ), store at -20 °C

  15. MycoZap plus-PR (Lonza, catalog number: VZA2021 ), store at -20 °C

  16. HEPES buffer (Stemcell, catalog number: 0 7200 ), store at 4 °C

  17. BrainPhysTM without phenol red medium (Stemcell, catalog number: 05791), store at 4 °C

  18. F(syn)W-RBN::VGLUT1-venus (PMID: 23581566, available upon request to Dr. Etienne Herzog), store at -80 °C

  19. F(syn)W-RBN::Synaptobervin2-EGFP (PMID: 23581566, available upon request to Dr. Etienne Herzog), store at -80 °C

  20. Complete DMEM medium (see Recipes)

  21. Complete Neurobasal A medium (see Recipes)

  22. Complete BrainPhys medium (see Recipes)

Equipment

  1. Leica DMI 6000 microscope (Leica Microsystems, Wetzlar, Germany)

  2. Spinning disk confocal head Yokogawa CSU-X1(Yokogawa Electric Corporation, Tokyo, Japan)

  3. EM-CCD QuantEM camera (Photmetrics, Tucson, USA)

  4. iLAS FRAP scanner system (Roper Scientific, Evry, France)

  5. Piezo nanofocusing scanner P721.LLQ (Physik Instrumente, Karlsruhe, Germany)

  6. Thermal incubator (Life Imaging Services, Switzerland)

Software

  1. MetaMorph microscopy automation and image analysis software (Molecular Devices, Sunnyvale, USA, https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy)

  2. ImageJ (NIH, USA, https://imagej.nih.gov/ij)

  3. FRAP Analysis Macro (Fabrice P. Cordelieres, https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM)

Procedure

  1. Prepare dissociated hippocampal neuronal culture for live imaging

    1. Coat the glass coverslips or glass bottom dishes with 0.1 mg/ml poly-L-lysine overnight, and rinse with ddH2O 3 times before using.

    2. Dissect hippocampi from P0 (Post-natal day 0) C57/BL6 mice in ice-cold Leibovitz’s L-15 medium.

    3. Collected all hippocampi in a 15 ml Falcon tube filled with ice-cold Leibovitz’s L-15 medium.

    4. Remove Leibovitz’s L-15 medium. Incubate tissues in 5 ml of 0.05% trypsin-EDTA solution at 37 °C for 15 min. Trypsin will digest extracellular proteins to facilitate cell dissociation.

    5. Remove 0.05% trypsin-EDTA solution, and replace with complete DMEM medium to stop the reaction.

    6. Then remove the DMEM medium, and wash the tissue with 5 ml complete Neurobasal A medium.

    7. Add 1 ml complete Neurobasal A medium in the tube. Dissociate cells mechanically by pipetting up and down with a 200 µl tip for 10-15 strokes.

    8. Incubate the cell suspension for 3 min to allow the sedimentation of large tissue debris. Take the 800 µl upper suspension and avoid sampling of tissue clusters.

    9. Measure cell density and plate cells to pre-coated poly-L-lysine glass coverslips/glass bottom dishes at a density of 20,000 cells/cm2.

      Note: Cells are grown in 2 ml complete Neurobasal A medium in the well of 12-well plate, or 3 ml medium in a 35 mm glass bottom dish.

    10. Cells are grown in complete Neurobasal A medium for 5 days in-vitro (DIV).

    11. From DIV 5-6, replace half conditioned medium (1 ml for each well of 12-well plate, and 1.5 ml for 35 mm glass bottom dishes) with complete BrainPhys medium every 2-3 days.


  2. Viral transduction of reporter gene

    On DIV 1 or 2, transduce cells with the lentivirus vector allowing for the expression of a fluorescent reporter of synaptic vesicles. We use F(syn)W-RBN::Synaptobrevin2-EGFP or F(syn)W-RBN::VGLUT1-venus. For each vector batch, Western-blot and fluorescent imaging are performed to adjust the dilution to limit protein overexpression to 2-fold of wild-type levels.

    The lenti vector dilution and protein expression are measured by Western-blot and fluorescent imaging:

    1. Cells are grown in 6 cm dishes that are pre-coated with poly-L-lysine with the same cell density as described above.

    2. On DIV 1 or 2, lentivirus (titer range in ~×108 TU/ml) is diluted to 1/100 with Neurobasal A medium. Add 20/40/80 µl diluted virus to each dish of the culture for transduction. Return the culture to the incubator.

    3. On DIV17, the cells are scraped and collected from the dishes. The targeted protein expression level in each sample is measured by Western-blot. VGLUT1-venus or Synaptobrevin2-EGFP has ~27 kDa higher molecular weight, thus can be easily distinguished from the corresponding endogenous wildtype protein. The increased virus transduction normally will result a linearized increase of protein expression.

    4. Compare the expression of VGLUT1-venus/Synaptobervin2-EGFP with the wildtype VGLUT1/Synaptovervin2. Calculate the right dilution of virus which results less than 2-fold of protein overexpression. Normally, the fluorescent intensity of the presynaptic boutons is bright enough and is suitable for FRAP imaging with this protein expression level.

    5. Based on the cell amount, proportionally add the right dilution of virus to the 12-well plates or 35 mm glass bottom dishes culture on DIV 1 or 2 that are prepared for FRAP imaging (Figure 2A).


  3. FRAP Imaging

    Note: FRAP imaging is performed with a spinning disk confocal microscope that is controlled by MetaMorph microscopy automation software. Take DIV 17-21 cell culture for imaging, normally neurons are mature and synaptic networks are well established at this time point. Incubate the cultures in conditioned culture medium (cells are grown in) containing HEPES (40 mM) and at physiological temperature (37 °C) during the imaging procedure.

    1. Use MetaMorph to control the entire FRAP procedure.

    2. Randomly select one field of the cell culture for imaging with a 63×/1.4 numerical aperture oil-immersion objective (Figure 2B).

    3. Select up to 5 fluorescent boutons that are distant from each other as Regions of Interest (ROIs) for bleaching (Figure 2B). Selecting too many boutons that are closely related on the same axon would affect the measurement. Before bleaching, monitor the boutons fluorescence every 30 s for 3 min (Figure 2A). Image as a Z-stack of 4.8 µm thickness with 0.8 µm step interval. ROIs should be at the midplane of the stack. Stack imaging allows for a more robust measurement of bouton intensity and to buffer fluctuations due to small Z-directed movements.

    4. Apply three passes of 491 nm laser (40 mW) to bleach the Syb2EGFP labeling boutons; two laser passes of 491 nm laser (30 mW) and the 405 nm laser (10 mW) to bleach the VGLUT1venus labeling boutons (Figure 2B). After bleaching, the fluorescence of ROIs should remain around 40%-60% of the initial fluorescence intensity (Figure 2C). VENUS and EYFP related dyes require a stimulation at 405 nm to prevent fluorescence recovery from a reversible dark state of the fluorescent proteins (McAnaney et al., 2005; Herzog et al., 2011).

    5. Monitor the fluorescence recovery after bleaching every 30 s during the first 3 min and then every 5 min during the next 70 min (Figure 2A).

Data analysis

  1. Open the stack sequence with ImageJ and perform a sum Z projection that will generate a 32 bits/pixel sequences (Video 1).

    Video 1. An example of image processing with ImageJ FRAP analysis Macro


  2. Use Image J FRAP Analysis Macro under “plugin” (https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM) to automate image analysis (Video 1).

    1. The macro commands apply x-y realignment to each individual stack.

    2. Then extract the integrated fluorescence intensity of bleached boutons, the cells in the field for photobleaching correction, and the background area for background subtraction (Figure 2E).

    3. The Macro will output all the raw data and processed data in a table named “Results” (Figure 3A) and a plot named “Values” that shows all ROIs’ intensity value (x stands for time point) as a function of time (Figure 3B).

  3. The boutons intensity slightly varies in each frame before bleaching. Take the average value before bleaching ()as prebleaching reference. Normalize boutons intensity at different time point  in the sequence as:

                        

  4. Filter out rejected ROIs from the final quantification which may correspond to the following cases:

    1. The bouton (ROI) fluorescence (b stands for the frame that right after bleaching) are over-bleached or less-bleached, which means < 20% or > 70% (Figure 3B, the bouton with black curve). Indeed,  below 20% the risk of photodamage to the sample is high, and above 70% the recovery will not be measured accurately.

    2. Because axonal exchange is not entirely linear and continuous, some boutons may receive or loose massive fluorescent clusters during the fluorescence recovery time (Figure 3B, the bouton with green curve). This was extensively described in our former paper (Herzog et al., 2011).

  5. Calculate the percentage of recovered fluorescence of each bouton. Normalize the boutons average intensity before bleaching as 1, right after bleaching as 0. The total bleached fluorescence is 1 - , the recovered fluorescence at different time point is .

                    

    The recovered fluorescent value  stands for the SVs exchange between presynaptic cluster and super-pool.

  6. Summarize the normalized bouton fluorescence data in your favorite software for statistics and plotting (Figure 3C). Fit the average of all normalized FRAP traces with a double exponential function (Figure 3D).



    Figure 3. FRAP Data analysis. A. When FRAP Analysis Macro analysis is done, a results file with the ROIs’ raw data and normalized data is generated. B. And a plot presenting the corrected normalized values as a function of time for all ROIs is generated. C. An example figure showed the summary of normalized FRAP traces. One colorful trace stands for an individual bouton fluorescence variation in the FRAP experiment. The black dots stand for the mean value of all the ROIs at different time points. D. A double exponential trace is fitted to the average FRAP curve.

Notes

  1. Use serum-free medium for mixed glial-neuronal culture. Because it is important to keep the cell morphology stable during the whole acquisition. Neurons grown on a layer of astrocytes will not be suitable for imaging, because of movements during the imaging procedure. Mixed culture containing few glia cells or banker culture are suitable for this long-time FRAP imaging.

  2. Select a field with nice neuronal network but with at least one empty region (no fluorescent material) for imaging. The dark region is essential to be taken as a background reference for later imaging analysis.

  3. Apply certain amount of 405 nm laser for Venus/EYFP fluorescence bleaching. As previously reported, YFP has a photochemical reversible dark state representing roughly 20% of YFP/VENUS molecules. This dark pool of fluorescent proteins is emptied by photoactivation at 405 nm (McAnaney et al., 2005; Herzog et al., 2011). Thus, accompanying bleaching with UV light significantly reduces the unwanted recovery.

  4. Set the right laser power for the bouton fluorescence bleaching. The bleach will result a ~50% fluorescence reduction. To avoid the bleaching variance caused by a single laser pass, repeat a mild laser pass for 2-3 times to the selected boutons is recommended. The appropriate laser power needs to be tested based on your own culture before experiments.

Recipes

  1. Complete DMEM medium

    450 ml DMEM medium

    50 ml FBS

    and 5 ml Penicillin-streptomycin

    store at 4 °C

  2. Complete Neurobasal A medium

    50 ml Neurobasal A medium

    1 ml B27 supplement

    125 µl Glutamax

    And 100 µl Mycozap plus-PR

    Store at 4 °C

  3. Complete BrainPhys medium

    50 ml BrainPhys medium

    1 ml B27 supplement

    125 µl Glutamax

    And 100 µl Mycozap plus-PR

    Store at 4 °C

Acknowledgments

We thank Bordeaux Imaging Center for their excellent technical support and equipment for the experiments. This work is supported by the Erasmus Mundus ENC program and the LabEx BRAIN extension grant (ANR-10-LABX-43 BRAIN), Agence Nationale de la Recherche (ANR-12-JSV4-0005-01 VGLUT-IQ ; ANR-10-LABX-43 BRAIN; ANR-10-IDEX-03-02 PEPS SV-PIT) and Fondation pour la Recherche Médicale (ING20150532192). This protocol introduced here was detailed from past studies (Herzog et al., 2011; Zhang et al., 2019).

Competing interests

The authors declare no financial or non-financial competing interests related to this work.

Ethics

The experimental design and all procedures were performed in accordance with the European guide for the care and use of laboratory animals and approved by the ethics committee of Bordeaux University (CE50) under the APAFIS n°1692.

References

  1. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. and Webb, W. W. (1976). Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16(9): 1055-1069.
  2. Denker, A. and Rizzoli, S. O. (2010). Synaptic vesicle pools: an update. Front Synaptic Neurosci 2: 135.
  3. Herzog, E., Nadrigny, F., Silm, K., Biesemann, C., Helling, I., Bersot, T., Steffens, H., Schwartzmann, R., Nagerl, U. V., El Mestikawy, S., Rhee, J., Kirchhoff, F. and Brose, N. (2011). In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice. J Neurosci 31(43): 15544-15559.
  4. Imig, C., Min, S. W., Krinner, S., Arancillo, M., Rosenmund, C., Sudhof, T. C., Rhee, J., Brose, N. and Cooper, B. H. (2014). The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84(2): 416-431.
  5. Korogod, N., Petersen, C. C. and Knott, G. W. (2015). Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation. Elife 4: e05793.
  6. Landis, D. M., Hall, A. K., Weinstein, L. A. and Reese, T. S. (1988). The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse. Neuron 1(3): 201-209.
  7. McAnaney, T. B., Zeng, W., Doe, C. F., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X., Boxer, S. G. and Bagshaw, C. R. (2005). Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. Biochemistry 44(14): 5510-5524.
  8. Qin, K., Sethi, P. R. and Lambert, N. A. (2008). Abundance and stability of complexes containing inactive G protein-coupled receptors and G proteins. FASEB J 22(8): 2920-2927.
  9. Sankaranarayanan, S., De Angelis, D., Rothman, J. E. and Ryan, T. A. (2000). The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79(4): 2199-2208.
  10. Siksou, L., Rostaing, P., Lechaire, J. P., Boudier, T., Ohtsuka, T., Fejtova, A., Kao, H. T., Greengard, P., Gundelfinger, E. D., Triller, A. and Marty, S. (2007). Three-dimensional architecture of presynaptic terminal cytomatrix. J Neurosci 27(26): 6868-6877.
  11. Soykan, T., Kaempf, N., Sakaba, T., Vollweiter, D., Goerdeler, F., Puchkov, D., Kononenko, N. L. and Haucke, V. (2017). Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly. Neuron 93(4): 854-866 e854.
  12. Staras, K., Branco, T., Burden, J. J., Pozo, K., Darcy, K., Marra, V., Ratnayaka, A. and Goda, Y. (2010). A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons. Neuron 66(1): 37-44.
  13. Südhof, T. C. (1995). The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375(6533): 645-653.
  14. Zhang, X. M., Francois, U., Silm, K., Angelo, M. F., Fernandez-Busch, M. V., Maged, M., Martin, C., Bernard, V., Cordelieres, F. P., Deshors, M., Pons, S., Maskos, U., Bemelmans, A. P., Wojcik, S. M., El Mestikawy, S., Humeau, Y. and Herzog, E. (2019). A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency. Elife 8: e50401.

简介

[摘要]突触小泡(SVs)聚集在突触前的末端,并沿轴突持续运输。根据其发布功能,SV分为不同的“池”。在多个突触前末端之间传播的SV的成像已帮助定义了一个名为“ SV超级池”的新池。在这里,我们描述了一种光漂白后的荧光恢复(FRAP)方法,以阐明超池中的SV与突触前末端的SV簇之间的关系。该方法对于研究SV迁移率调节机制非常有效。


[背景]突触小泡(SVs)是通过神经递质的储存和释放参与神经传递的关键细胞器。SV大多在邻近突触前末端活动区的簇中识别。在电子显微镜(EM)下,SV具有直径为40-50nm的均匀外观(Landis等,1988; Korogod等,2015)。据我们所知,SV之间没有明显的生化区别。在不同的刺激范式下,它们显示出不同的释放特性。因此,SV被分为不同的功能池:储备池,回收池和易于释放池(图1)(Denker和Rozzoli,2010)。EM深入研究了详细的突触结构,SV定位,SV释放机制。发现SV通过细丝与一个或两个相邻的囊泡相连,突触素被认为是连接器的一部分,并且将SV保持在储备池中(Siksou et al。,2007)。超结构研究也揭示了SV对接和融合的分子步骤的解剖(Imig等人,2014)。SV与质膜的融合将酸性管腔(pH约为5 .0 )暴露于中性细胞外介质(pH值为7.4)(Südhof,1995)。因此,还通过用pH传感器标记SV来深入研究SV的循环利用和相关的分子机制(Sankaranarayanan等,2000; Soykan等,2017)。

图URE 1.突触小泡池。突触小泡(SV)池的经典模型是储备池,回收池和易于释放的池。新定义的“超级池”包括SV的那间共享连接-passant沿轴突突触前终扣。

多年来,人们认为的SV回收单个突触前终端内无重大与邻居交换连接-passant相同轴突终扣,W往往微不足道终端可以与来自遥远的胞体显著自治功能。在过去的十年中,SV的观察贩运和多间共享连接-passant沿轴突突触前终扣。该轴突SV种群被称为“超级池”(图1)(Denker和Rozzoli,2010)。通过体外和体内范例观察到了该超级池(Staras等,2010; Herzog等,2011; Zhang等,2019)。然而,在超级池中的SV如何促进神经传递仍然是未知的。我们最近可以显示出,超级水池尺寸的变化会影响自发释放频率(Zhang et al。,2019)。

FRAP通常用于确定细胞膜扩散或蛋白质结合的动力学(Axelrod等,1976; Qin等,2008),而SV迁移的动力学与这些情况不同。但是,如上所述,一定数量的SV聚集在每个突触前按钮中,并且在超级池中始终用SV刷新。FRAP允许测量SV在突触前钮扣和轴突之间的扩散。在这里,我们详细介绍了FRAP成像的协议,它能够阐明交流的超级游泳池和突触前SV集群之间的SV。SVs用标记有荧光蛋白的突触小泡蛋白标记,例如VGLUT1金星或Synaptobrevin 2 EGFP (图2A-2D)。这种FRAP方法可以应用于体外和体内系统。在本文中,我们将描述应用于离体海马神经元培养的详细协议。这种FRAP成像方法将有助于进一步探索与SV迁移率相关的机制。

图2. FRAP序列采集和分析。在神经元中,SV被荧光蛋白Venus / EGFP标记。突触前的按钮包含一簇SV,因此显示为明亮的单个按钮。A.实验工作流程。在培养物的DIV 1/2上进行病毒转导,从DIV 17到21获取FRAP成像。详细的FRAP成像过程显示在黄色框中。B.随机选择一个培养区域进行成像。在该字段中,选择5个彼此相距较远的boutons(标记为红色圆圈)作为用于漂白的ROI。C.刚漂白后就获得的框架。由于光漂白,5个选定的按钮的荧光强度显着降低。D.光漂白1小时后获得的框架,ROI的荧光强度逐渐恢复。E.用FRAP分析宏分析FRAP序列。在弹出窗口中的菜单上,选择漂白钮扣的区域(红色圆圈),用于光漂白校正的字段中的单元格(黄色区域)和用于背景扣除的背景区域(蓝色区域)。

关键字:突触泡, 膜运输, 活细胞成像, 光漂白后的荧光恢复技术, 轴突运输, 突触

材料和试剂
圆形玻璃盖,#1厚度,18毫米(Warner Instruments,目录号:64-0384)
35毫米玻璃底盘,带有10毫米微孔(Cellvis ,目录号:D35-10-1-N)
15 ml锥形离心管(Falcon TM ,目录号:14-959-53A)
200 µl移液器吸头(QuickRack ,目录号:NC9640144)
C57 / BL6J小鼠(杰克逊实验室,目录号:000664)
莱博维茨的L-15培养基(Gibco ,目录号:11415064),在4 °C下储存
0.05%胰蛋白酶-EDTA(Gibco ,目录号:25300054),储存在-20°C
Dulbecco的改良Eagle's培养基(DMEM)(Gibco ,目录号61965026),在4°C下储存
胎牛血清(Eurobio ,目录号:CVFSVF001),储存在-20°C
青霉素链霉素(Gibco ,目录号:15140122),储存在-20°C
聚-L-赖氨酸(Sigma,目录号:P2636),储存在-20°C
Neurobasal A培养基(Gibco ,目录号:12349105),储存在4°C
B27补充剂(Gibco ,目录号:17504044),储存在-20°C
Glutamax (Gibco ,目录号:35050038),储存在-20°C
MycoZap plus-PR(Lonza ,目录号:VZA2021),储存在-20°C
HEPES缓冲液(干细胞,目录号:07200),存储在4°C
不含酚红培养基(Stemcell ,目录号:05791)的BrainPhys TM ,储存在4°C下
F(syn )W-RBN :: VGLUT1-维纳斯(PMID:23581566,可应要求向Etienne Herzog博士提供),储存于-80 °C
F(syn )W-RBN :: Synaptobervin2-EGFP(PMID:23581566,可应要求提供给Etienne Herzog博士),储存于-80 °C
完整的DMEM介质(请参阅食谱)
              完整的神经基础A培养基(请参阅食谱)
完整的BrainPhys培养基(请参阅食谱)


设备


Leica DMI 6000显微镜(德国Wetzlar的Leica Microsystems )
旋转盘共焦头横河电机CSU-X1(横河电机公司,日本东京)
EM-CCD QuantEM相机(Photmetrics ,美国图森)
iLAS FRAP扫描仪系统(法国埃夫里Roper Scientific )
压电纳米聚焦扫描仪P721.LLQ(Physik Instrumente ,德国卡尔斯鲁厄)
热培养箱(瑞士生命成像服务)

软件
MetaMorph显微镜自动化和图像分析软件(Molecular Devices,美国桑尼维尔,https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy)
ImageJ(美国国立卫生研究院,https://imagej.nih.gov/ij)
FRAP分析宏(Fabrice P.Cordelieres ,https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM)


程序


准备分离的海马神经元培养物以进行实时成像
在玻璃盖玻片或玻璃底盘上涂0.1 mg / ml聚L-赖氨酸过夜,并在使用前用ddH 2 O冲洗3次。
在冰冷的Leibovitz的L-15培养基中,从P0(产后第0天)C57 / BL6小鼠中解剖海马。
将所有海马收集在装满冰冷的莱博维茨L-15培养基的15毫升猎鹰管中。
移除Leibovitz的L-15培养基。在5毫升孵育组织的0.05%胰蛋白酶-EDTA溶液在37℃下15分钟。胰蛋白酶将消化细胞外蛋白以促进细胞解离。
除去0.05%的胰蛋白酶-EDTA溶液,并用完全的DMEM培养基代替以终止反应。
然后取出DMEM培养基,并用5 ml完整的Neurobasal A培养基洗涤组织。
在试管中加入1 ml完整的Neurobasal A培养基。用200 µl吸头上下吸打10-15次,以机械方式解离细胞。
孵育细胞悬浮液3分钟,以使大的组织碎片沉淀下来。取800 µl上悬液,避免取样组织簇。
测量细胞密度并将平板细胞以20,000个细胞/ cm 2的密度涂到预涂的聚L-赖氨酸玻璃盖玻片/玻璃底盘上。
注意:细胞在12孔板孔中的2 ml完整Neurobasal A培养基中或在35 mm玻璃底皿中的3 ml培养基中生长。


细胞在完全Neurobasal A培养基中体外培养5天(DIV)。
从DIV 5-6,每2-3天用完全的BrainPhys培养基替换一半条件培养基(12孔板的每个孔1 ml,35 mm玻璃底皿1.5 ml)。


报道基因的病毒转导
在DIV 1或2上,用慢病毒载体转导细胞,以表达突触小泡的荧光报告分子。我们使用F(syn )W-RBN :: Synaptobrevin2-EGFP或F(syn )W-RBN :: VGLUT1-venus。对于每批载体,进行蛋白质印迹和荧光成像以调节稀释度以将蛋白质过表达限制为野生型水平的2倍。


所述LENTI载体稀释和蛋白质表达通过Western印迹和荧光成像测定:


细胞在6厘米培养皿中生长,该培养皿预涂有聚L-赖氨酸,具有与上述相同的细胞密度。
在DIV 1或2上,用Neurobasal A培养基将慢病毒(滴度范围在〜× 1 0 8 TU / ml中)稀释至1/100 。20/40/80添加μ l的稀释病毒到培养转导的每个菜肴。将文化传回培养箱。
在DIV17上,将细胞刮下并从培养皿中收集。通过蛋白质印迹法测量每个样品中的靶向蛋白质表达水平。VGLUT1-维纳斯或Synaptobrevin2-EGFP具有〜27 kD的较高分子量,因此可以容易地与相应的内源性野生型蛋白区分开。病毒转导的增加通常会导致蛋白质表达的线性增加。
比较表达的用野生型VGLUT1 / Synaptovervin2 VGLUT1金星/ Synaptobervin2-EGFP。计算正确的病毒稀释度,使蛋白质过表达少于2倍。通常,突触前钮扣的荧光强度足够明亮,适合具有此蛋白表达水平的FRAP成像。
根据细胞数量,按比例将正确稀释的病毒稀释液添加到DIV 1或2上的12孔板或35 mm玻璃底培养皿中,以进行FRAP成像(图2A)。


FRAP成像
注意:FRAP成像是由MetaMorph显微镜自动化软件控制的旋转盘共聚焦显微镜进行的。采取DIV 17 - 21细胞培养物用于成像,通常神经元的成熟和突触网络在该时间点很好地建立。在成像过程中,在含有HEPES(40 mM )的条件培养基(细胞在其中生长)和生理温度(37 °C)下孵育培养物。


使用MetaMorph来控制整个FRAP过程。
随机选择一个细胞培养场,以63 × /1.4数值孔径油浸物镜成像(图2B)。
选择最多5个彼此远离的荧光按键作为感兴趣区域(ROI)进行漂白(图2B)。选择太多在同一个轴突上紧密相关的钮扣将影响测量。在漂白之前,每30秒钟监测3次布顿荧光3分钟(图2A)。图像为厚度为4.8 µm的Z堆栈,步距为0.8 µm。ROI应位于堆栈的中平面。叠层成像可以更稳定地测量钮扣强度,并可以缓冲由于Z方向较小的运动而引起的波动。
应用三遍491 nm激光(40 mW )漂白Syb2 EGFP标记钮扣;两次491 nm激光(30 mW )和405 nm激光(10 mW )激光通过漂白VGLUT1金星标记钮扣(图2B)。漂白后,ROI的荧光应保持在初始荧光强度的40%-60%左右(图2C)。VENUS和EYFP相关的染料需要在405 nm处进行刺激,以防止荧光蛋白从可逆的暗态恢复到荧光状态(McAnaney等,2005; Herzog等,2011;)。
在最初的3分钟内每30秒漂白一次,然后在接下来的70分钟内每5分钟漂白一次,监测荧光恢复(图2A)。                           


数据分析


使用Imag eJ打开堆栈序列,并执行Z总和投影,这将生成32位/像素序列(视频1)。




视频1.使用ImageJ FRAP分析宏进行图像处理的示例


使用“插件”(https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM)下的Image J FRAP分析宏来自动进行图像分析(视频1)。
宏命令将xy重新对齐应用于每个单独的堆栈。
然后提取积分的荧光强度的漂白的boutons,在现场的细胞用于光漂白校正和背景区域的背景扣除(图2E)。
宏将在名为“结果”的表(图3A)和名为“值”的图中输出所有原始数据和处理后的数据,该图显示了所有ROI强度值(代表时间点)随时间的变化(图3B) )。 
在漂白之前,每一帧的钮扣强度都略有变化。取漂白前的平均值(作为漂白前的参考。将序列中不同时间点的布顿强度归一化为:






从最终定量中筛选出拒绝的ROI,这可能与以下情况相对应:
bouton(ROI)荧光(代表漂白后立即显示的框架)被过度漂白或漂白程度较低,这意味着<20%或> 70%(图3B,带有黑色曲线的bouton)。的确,低于20%对样品产生光损坏的风险很高,而高于70%则无法准确测量回收率。
由于轴突交换不完全是线性且连续的,因此在荧光恢复时间内,某些钮扣可能会接收或散布大量的荧光团(图3B,绿色曲线的钮扣)。在我们以前的论文中对此进行了广泛描述(Herzog等,2011)。
计算每个按钮的荧光恢复百分比。将漂白前的布顿平均强度归一化为1,将漂白后的刚度平均强度归一为0。总漂白荧光为,在不同时间点恢复的荧光为。






恢复的荧光值代表突触前簇和超级池之间的SV交换。


在您喜欢的软件中汇总归一化的bouton荧光数据,以进行统计和绘图(图3C)。用双指数函数拟合所有归一化FRAP迹线的平均值(图3D)。




图3. FRAP数据分析。A.完成FRAP分析宏分析后,将生成一个包含ROI原始数据和规范化数据的结果文件。B.生成了一个曲线图,该曲线图显示了所有ROI的校正归一化值与时间的关系。C.示例图显示了归一化FRAP迹线的摘要。一条彩色迹线代表FRAP实验中的单个bouton荧光变化。黑点代表在不同时间点所有ROI的平均值。D.双指数迹线拟合到平均FRAP曲线。


笔记


使用无血清培养基进行神经胶质-神经混合培养。因为在整个采集过程中保持细胞形态稳定很重要。由于在成像过程中的运动,在星形胶质细胞层上生长的神经元将不适合成像。含有少量神经胶质细胞的混合培养物或banker培养物适用于这种长时间的FRAP成像。
选择一个具有良好神经网络但至少有一个空白区域(无荧光物质)的成像区域。暗区对于以后的成像分析必不可少。
应用一定量的405 nm激光进行Venus / EYFP荧光漂白。如先前报道,YFP具有光化学可逆的暗态,约占YFP / VENUS分子的20%。荧光蛋白的这个暗池通过在405 nm处的光激活被清空(McAnaney et al。,2005; Herzog et al。,2011)。因此,伴随紫外线的漂白显着降低了不希望的回收率。
小号等为布顿荧光漂白右激光功率。漂白剂将导致荧光减少约50%。为避免单次激光通行引起的漂白变化,建议对选定的钮扣重复2-3次轻柔的激光通行。在实验之前,需要根据您自己的文化来测试适当的激光功率。


菜谱


              完整的DMEM介质
450 ml DMEM培养基


50毫升FBS


和5 ml青霉素链霉素


储存在4°C


              完整的神经基础A培养基
50 ml神经基础A培养基


1毫升B27补品


125 µl的Glutamax


和100 µl Mycozap plus-PR


储存在4°C


完整的BrainPhys培养基
50毫升BrainPhys培养基


1毫升B27补品


125 µl的Glutamax


和100 µl Mycozap plus-PR


储存在4°C


致谢


我们感谢波尔多影像中心的出色技术支持和实验设备。这个工作是由伊拉斯谟ENC方案和支持LabEx BRAIN扩展许可(ANR-10的LabX-43 BRAIN),法新社国立德拉RECHERCHE (ANR-12-JSV4-0005-01 VGLUT- IQ; ANR -10-的LabX-43脑; ANR-10-IDEX-03-02 PEPS SV-PIT)和基金会倒拉RECHERCHE MEDICALE (ING20150532192)。从过去的研究中详细介绍了此处引入的该协议(Herzog等,2011; Zhang等,2019)。


利益争夺


作者声明没有与此工作相关的财务或非财务竞争利益。




伦理


实验设计和所有程序均按照欧洲实验动物的护理和使用指南进行,并由波尔多大学伦理委员会(CE50)批准,依据APAFIS n°1692。


参考


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Copyright Zhang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, X. M., Cordelières, F. and Herzog, E. (2021). Investigate Synaptic Vesicles Mobility in Neuronal Culture Axons by FRAP Imaging. Bio-protocol 11(6): e3962. DOI: 10.21769/BioProtoc.3962.
  2. Zhang, X. M., Francois, U., Silm, K., Angelo, M. F., Fernandez-Busch, M. V., Maged, M., Martin, C., Bernard, V., Cordelieres, F. P., Deshors, M., Pons, S., Maskos, U., Bemelmans, A. P., Wojcik, S. M., El Mestikawy, S., Humeau, Y. and Herzog, E. (2019). A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency. Elife 8: e50401.
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