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FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction
果蝇神经肌肉接头处FM1-43光转换和电子显微镜分析   

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Abstract

We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs.

Keywords: Electron microscopy(电子显微镜检查), Photoconversion(光转换), FM1-43(FM1-43), Drosophila(果蝇), Synaptic vesicle(突触小泡), Recycling pool(回收池), Neuromuscular junction(神经肌肉接头)

Background

Neuronal transmitters are released via the fusion of synaptic vesicles with the neuronal plasma membrane. Vesicles can fuse spontaneously or in response to an action potential. Subsequently, vesicles become retrieved via endocytosis and recycled. Molecular mechanisms of synaptic vesicle recycling were investigated extensively with the tools of molecular biology, electrophysiology and microscopy (Slepnev and De Camilli, 2000; Sudhof, 2004; Rizzoli and Betz, 2005; Kavalali, 2006). Loading the endocytic marker FM1-43 coupled with the dye photoconversion followed by electron microscopy analysis is a powerful technique that allows the investigation and measurement of the recycling vesicle pools (Harata et al., 2001; Schikorski and Stevens, 2001; Rizzoli and Betz, 2004). Drosophila neuromuscular junction (NMJ) is an advantageous preparation with clearly defined synaptic boutons, which enables rapid generation of lines with mutated synaptic proteins and rigorous evaluation of vesicle recycling pools (Akbergenova and Bykhovskaia, 2009; Denker et al., 2009). A fundamental question in the field of synaptic transmission is whether the evoked and spontaneous transmission utilizes the same recycling pool. To address this question, the recycling pool utilized in the absence of stimulation needs to be measured. This is a challenging problem due to low rates of spontaneous release and recycling. We have developed a protocol for FM1-43 loading followed by the dye photoconversion and EM analysis, which enables rigorous evaluation of recycling pools utilized during spontaneous and evoked transmission at the Drosophila NMJ (Sabeva et al., 2017).

Materials and Reagents

  1. Sample preparation
    1. Gloves
    2. Long sleeve lab coat
    3. Sample bottle with snap cap, size 4 ml (Electron Microscopy Sciences, catalog number: 64250 )
    4. Falcon 35 mm culture plates (Corning, NY)
    5. Drosophila melanogaster fly stocks: Canton S (Bloomington Drosophila Stock Center, catalog ID: FBst1000081) and cpxSH1 (cpx-/-) (Huntwork and Littleton, 2007)
    6. Ca2+-free HL3 saline containing 75 μM advasep-7 (Biotium, catalog number: 70029 ) (see Note 1)
    7. FM1-43 (Thermo Fisher Scientific, InvitrogenTM, catalog number: T35356 ), 10 µM
    8. 100 mM NH4Cl (Sigma-Aldrich, catalog number: A9434 ) in HEPES
    9. 1.5 mg/ml DAB (Agilent Technologies, DAKO, OEM) in HEPES
      Note: The compound is available liquid or tablets in kits.
    10. 1% osmium tetroxide (OsO4) prepared in 90 mM cacodylate buffer using the 4% OsO4 stock solution (Electron Microscopy Sciences, catalog number: 19150 )
    11. Ascending acetone series 50, 70, and 90% prepared from 100%
    12. 2% uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 ) prepared in water
    13. Acetone, 100% (Sigma-Aldrich, catalog number: 270725 ) dehydrated with molecular sieve dehydrate Fluka (Fluka Analysis, Sigma-Aldrich, catalog number: 270725 )
    14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
    15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
    16. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
    17. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
    18. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014 )
    19. Trehalose (Sigma-Aldrich, catalog number: T9531 )
    20. Sucrose (Sigma-Aldrich, catalog number: S1888 )
    21. HEPES-Na salt (Sigma-Aldrich, catalog number: H7006 )
    22. HEPES (Sigma-Aldrich, catalog number: H3375 )
    23. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
    24. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220 )
    25. Sodium cacodylate buffer (Electron Microscopy Sciences, catalog number: 11653 )
    26. HL3 Drosophila saline solution (see Recipes)
    27. Pre-fixative solution (see Recipes and Note 2)
    28. HEPES-buffered saline (see Recipes)
    29. Fixative solution (see Recipes and Note 2)

  2. Sample embedding
    1. ACLAR® 33C Embedding film (Electron Microscopy Sciences, catalog number: 50425-10 )
    2. Tri-Corn beakers: plastic, disposable (Electron Microscopy Sciences, catalog number: 60972 )
    3. Flat Bottom Embedding Capsules (Electron Microscopy Sciences, catalog number: 70021 )
    4. Epon (Embed-812) (Electron Microscopy Sciences, catalog number: 14900 )
    5. Nadic Methyl Anhydride (NMA) (Electron Microscopy Sciences, catalog number: 19000 )
    6. Dodecenyl Succinic Anhydride Specially Distilled (DDSA) (Electron Microscopy Sciences, catalog number: 13710 )
    7. 2,4,6-Tri(dimethylaminomethyl) phenol (DMP-30) (Electron Microscopy Sciences, catalog number: 13600 )
    8. ERL-4221 (Electron Microscopy Sciences, catalog number: 15004 )
    9. D.E.R. 736 Epoxy Resin (Used to simplify infiltration in combination with Embed 812) (Electron Microscopy Sciences, catalog number: 13000 )
    10. Nonenyl Succinic Anhydride Modified (NSA) (Electron Microscopy Sciences, catalog number: 19050 )
    11. Dimethylaminoethanol (DMAE) (Electron Microscopy Sciences, catalog number: 13300 )
    12. Embedding Mix A (see Recipes)
    13. Embedding Mix B (see Recipes)

Equipment

  1. Sample preparation
    1. Sylgard 184 (World Precision Instruments, Sarasota, FL)
    2. Fine tweezers (World Precision Instruments, models: #2 and #5 )
    3. Fine scissors (World Precision Instruments, model: 501233 )
    4. Insect Minutien Pins (0.1 mm) (Fine Science Tools, catalog number: 26002-10 )
    5. Dissecting stereoscopic zoom microscope (Nikon Stereozoom Microscope, Nikon Corporation)
    6. A.M.P.I. Master 8 Stimulator (A.M.P.I., model: Master-8 )
    7. Suction electrode filled with HL3
    8. Epifluorescence compound microscope with a filter cube with a long-pass emission filter customized for FM1-43 dye
    9. 60x water-immersion objective (ZEISS, Thornwood, NY)
    10. Mercury lamp
    11. Biowave (Ted Pella, Redding, CA)
    12. 480 ± 10 bandpass excision filter
    13. PELCO® R1 Single Speed Rotator in the 35° positions (Ted Pella, model: PELCO® R1 )
    14. Fume hood

  2. Sample embedding
    1. Oven Binder (Tuttlingen, Germany)

  3. Ultrathin Sections preparation and Image Capture
    1. Ultramicrotome for ultrathin sectioning (Leica, model: Leica EM UC6 )
    2. Formvar/carbon-coated 2 x 1 mm slot copper grids (Electron Microscopy Sciences, catalog number: FCF-2010-Cu )
    3. Ultra Diatome diamond sectioning knife, wet (Electron Microscopy Sciences, catalog number: 27-US )
    4. UltraTrim Dry Room Temperature Diatome knife, 4.0 mm (Electron Microscopy Sciences, catalog number: UTT-40-R )
    5. Eyelash with handle (Ted Pella, catalog number: 119 )
    6. Double edge stainless steel razor blades (Electron Microscopy Sciences, Hatfield, PA)
    7. Forceps for grids (Electron Microscopy Sciences, Hatfield, PA)
    8. JEOL 100 CX Electron Microscope equipped with Hamamatsu digital camera and AMT software

Software

  1. ImageJ (National Institutes of Health) for image analysis
  2. Adobe Photoshop (Adobe Systems) software for image analysis

Procedure

  1. Dissection of Drosophila melanogaster larvae in HL3 solution
    1. Select third-instar larvae (L3) and place it in a Sylgard dish.
    2. Pin the L3 on the anterior (head/mouth apparatus) and the posterior part, and the dorsal side-up.
    3. Immerse the pinched larvae in 4-5 ml HL3 solution (Recipe 1) to the Sylgard dish.
    4. Section the dorsal side longitudinally and remove the internal organs (intestines, fat bodies and trachea).
    5. Stretch and pin the rest of the L3.
    6. Several muscles could be used. In this study experiments were performed at Ib boutons in muscles 6 and 7. (Figure 1A)


      Figure 1. Third-instar larvae dissection. A. High magnification image of muscle fibers 7, 6, 13, 12 and V-shaped; B. Image of the ventral muscles in L3 where a nerve (black arrowhead) is sucked into the suction electrode connected to a stimulator; C and D. Adjusting the illumination times of the pre-fixed sample loaded with FM1-43 (WT larvae loaded for 30 sec). C. The DIC image shows that the illumination time of 9 min produces a successful photoconversion. Synaptic boutons can be identified as dark spots barely visible in the surface of the muscle (white arrows). D. The illumination time of 15 min is excessive, as evident from strong DAB precipitation, observed as large dark spots over the length of the NMJ (white arrows). Scale bar = 10 µm.

  2. Activity dependent labeling of Synaptic Vesicles with FM1-43 dye
    Note: Perform the following steps in low light conditions to protect the fluorophore component of FM1-43 dye. Cover the FM1-43 dye with foil or work in the dark at room temperature.
    1. Replace HL3 solution with HL3 containing 10 µM FM1-43.
    2. For loading in the absence of stimulation (spontaneous recycling pool) leave the preparation for the time of loading (10, 30, 120, and 600 sec were used in our studies).
    3. For active loading, using the head stage controllers pull the nerve into a suction electrode connected to a stimulator. Suck the end of the nerve. Use the stimulation protocol for low-frequency stimulation, 5 min, 5 Hz. Quickly remove the FM1-43 dye once the stimulation is complete (Figure 1B). The technique as adopted from Verstreken et al., 2008.
    4. Upon dye loading, rapidly wash the preparations three times for 30 sec in Ca2+-free HL3 saline containing 75 µM Advasep-7.

  3. Fixation
    1. Prefix the preparation (Recipe 2) for 15 min at room temperature.
    2. Wash three times for 5 min in HEPES buffer saline (Recipe 3).
    3. Leave the preparation covered in 100 mM NH4Cl for 10 min to quench autofluorescence of the fixative.
    4. Wash out the NH4Cl solution with HEPES buffer 2 times for 5 min.

  4. FM1-43 photoconversion
    1. Preincubate the preparation for 10 min in HEPES buffer containing 1.5 mg/ml DAB.
    2. Place the sample under an epifluorescent microscope. Identify the area of interest under 60x water immersion objective, at room temperature.
    3. Illuminate the sample for 8-10 min using a mercury lamp with a 480 ± 10 bandpass excision filter using the maximum light intensity. The dye will bleach with time. Observe the process of photoconversion by switching periodically to the bright field. As photoconversion takes place, dark brown DAB precipitate localizes at the illuminated area. The illumination time needs to be adjusted, as illustrated in Figures 1C and 1D.

  5. Post-fixation
    1. Wash the sample in HEPES three times 5 min.
    2. Immerse the preparation in the fixative solution (Recipe 4) and fix the sample in BioWave for 2 min at 100 W. Place the temperature insert close to the preparation to monitor temperature fluctuations. Do not allow the temperature to exceed 25-26 °C.
    3. Unpin the preparation carefully from the Sylgard dish. Transfer the preparation into a sample bottle with a snap cap.
    4. Wash the sample in 90 mM sodium cacodylate buffer, pH 7.4 twice for 5 min.

  6. EM sample processing
    Note: The following steps (unless specified) are performed at room temperature.
    1. Osmium tetroxide post-fixation and uranyl acetate staining
      1. Incubate the preparation in 1% OsO4 solution for 1 h at room temperature. The unused solution can be stored refrigerated for up to 1 month. Perform all steps under a fume hood, using gloves and long sleeve lab coat. Use 500 µl of this solution to immerse the sample. (Note 4)
      2. Wash with 90 mM cacodylate buffer for 5 min.
      3. Wash with deionized water for 5 min.
      4. Incubate the preparation in 2% uranyl acetate for 30-60 min. The unused solution can be stored refrigerated for up to 2 weeks.
      5. Wash with deionized water for 5 min twice.
    2. Dehydration
      1. Prepare separate solutions containing 50, 70, 90, and 100% of acetone.
      2. Add 1 ml of 50% acetone to the sample for 10 min.
      3. Add 1 ml of 70% acetone to the sample for 10 min.
      4. Add 1 ml of 90% acetone to the sample for 10 min.
      5. Add 1 ml of 100% acetone to the sample twice for 10 min.
    3. Embedding
      1. Mix 2 volumes of embedding resin (Note 3) with one volume of 100% acetone. Add it to the preparation under continuous rotation for 1 h or overnight.
      2. Keep the sample in embedding resin for at least 18 h (overnight).
      3. Under a stereomicroscope, place the preparation over ACLAR film, and then identify and trim the area of interest.
      4. Place the preparation over a new ACLAR film with the muscles facing down and the cuticle facing up. Position an embedding capsule filled with polymerized resin upside-down on the top of the specimen. Polymerize at 60 °C for 24-36 h.
      5. Remove the ACLAR film. This will leave sample attached to the embedding capsule.
    4. Thin sectioning and EM imaging
      1. Remove the block from the embedding capsule and mount the block into a specimen holder of an ultramicrotome.
      2. Under a stereomicroscope, mark the area of interest with a razor blade. The marked area should be a trapezoid that would fit onto the slot grid.
      3. Trim the block over the outlined area.
      4. Insert specimen holder with the block in the ultramicrotome and align the long edge of the trapezoid to the trimming knife edge. Trim 15-20 µm and shift the knife to trim the second long edge of the trapezoid. Trim another 15-20 µm. Repeat the same for the short sides of the trapezoid (Figure 2 and Videos 1-5).
      5. Align the block face exactly parallel to the edge of the diamond sectioning knife.
      6. Cut thin sections (50-60 nm).
      7. Collect individual sections (or collect sections in series) and place them on the 2 x 1 mm slot grids.
      8. Allow the sections to dry before taking images.
      9. Image the sections using conventional TEM.


        Figure 2. Sample block fine trimming. In the fixed and flat embedded Drosophila larvae the region of interest, muscles 6 and 7 are outlined with razor blade. Upon rough block trimming with razor blade and elimination of resin the face of the block is shaped with trimming knife. A. Fine trimming. The edge of the knife is aligned to the trapezoid top. B. Vertically positioned trapezoid with the left edge of the trimming knife aligned to the top of the trapezoid. C. Fine trimmed trapezoid with parallel top and bottom, and slightly tilted sides.
        Note: The paralleled top and bottom are important for forming a ribbon during serial sectioning.

        Video 1. Rough trapezoid trimming

        Video 2. Fine trapezoid trimming, part one. Trimming trapezoid top.

        Video 3. Fine trapezoid trimming, part 2. Once sectioned the trapezoid top the trimming knife is retracted and positioned to trapezoid bottom without additional alignment.

        Video 4. Fine trapezoid trimming, part 3. Trimming trapezoid bottom.

        Video 5. Serial sectioning

Data analysis

  1. Employing the photoconversion procedure, we investigated the recycling vesicle pool in the Drosophila larvae lacking the presynaptic protein complexin (cpx-/-) (Huntwork and Figure 3A), which enables the elevated rate of spontaneous release. In contrast, the recycling pool observed in WT boutons during spontaneous loading was negligibly small (data not shown, see Sabeva et al., 2017). In cpx -/-preparations loaded during the nerve stimulation, the recycling pool was also increased compared to WT preparations (Figures 3A and 3B), suggesting that separate recycling pools are utilized for the spontaneous and evoked release modes (Sabeva et al., 2017).


    Figure 3. Representative micrographs showing the recycling vesicle pools. A. cpx-/- boutons loaded in the absence of stimulation for 10, 30 or 120 sec. B. WT and cpx-/- boutons loaded during the nerve stimulation at 5 Hz for 5 min. Photoconverted labelled vesicles (black), non-labelled vesicles (grey). Scale bar = 500 nm.

  2. Detailed data processing analysis and replicates including applied statistical tests could be found in the original manuscript, see Sabeva et al., 2017.

Notes

  1. Advasep-7 is a β-cyclodextran derivative. It works as a dye scavenger and reduces background fluorescence.
  2. 16% paraformaldehyde solution and 25% glutaraldehyde solution can be purchased in 10 ml ampoules.
  3. Embedding resin: Prepare in a new disposable 250 ml beaker by mixing equal volumes of Embedding mix A (Recipe 5) and Embedding mix B (Recipe 6). Use a wooden tongue depressor to mix thoroughly. Degas (eliminate the air bubbles) for 5 min in a vacuum desiccator or leave 30 min at room temperature. Store tightly sealed at room temperature and use at the same day or aliquot and store at -20 °C.
  4. All steps during EM sample processing are performed in shaker or sample rotator.

Recipes

  1. HL3 Drosophila saline solution


  2. Pre-fixative solution


  3. HEPES-buffered saline


  4. Fixative solution


  5. Embedding mix A
    Prepare by mixing:
    20 g EMBed-812
    11 g DDSA
    9 g NMA
    0.8 ml DMP-30
    In 250 ml Tri-Corn disposable beaker
    Use a wooden tongue depressor to mix thoroughly
  6. Embedding mix B
    Prepare by mixing:
    10 g ERL
    6 g DER
    26 g NSA
    0.4 ml DMAE
    In 250 ml Tri-Corn disposable beaker
    Use a wooden tongue depressor to mix thoroughly

Acknowledgments

This work was supported by National Institutes of Health Grant R01 MH099557 and NIH NINDS SNRP Grant 5U54NS083924.

References

  1. Akbergenova, Y. and Bykhovskaia, M. (2009). Enhancement of the endosomal endocytic pathway increases quantal size. Mol Cell Neurosci 40(2): 199-206.
  2. Denker, A., Krohnert, K. and Rizzoli, S. O. (2009). Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction. J Physiol 587(Pt 12): 2919-2926.
  3. Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J. and Tsien, R. W. (2001). Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc Natl Acad Sci U S A 98(22): 12748-12753.
  4. Huntwork, S. and Littleton, J. T. (2007). A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat Neurosci 10(10): 1235-1237.
  5. Kavalali, E. T. (2006). Synaptic vesicle reuse and its implications. Neuroscientist 12(1): 57-66.
  6. Rizzoli, S. O. and Betz, W. J. (2004). The structural organization of the readily releasable pool of synaptic vesicles. Science 303(5666): 2037-2039.
  7. Rizzoli, S. O. and Betz, W. J. (2005). Synaptic vesicle pools. Nat Rev Neurosci 6(1): 57-69.
  8. Sabeva, N., Cho, W. R., Vasin, A., Gonzalez, A., Littleton, T. J. and Bykhovskaia, Maria. (2017). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci 37: 383-396.
  9. Schikorski, T. and Stevens, C. F. (2001). Morphological correlates of functionally defined synaptic vesicle populations. Nat Neurosci 4(4): 391-395.
  10. Slepnev, V. I. and De Camilli, P. (2000). Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci 1(3): 161-172.
  11. Sudhof, T. C. (2004). The synaptic vesicle cycle. Annu Rev Neurosci 27: 509-547.
  12. Verstreken, P., Ohyama, T. and Bellen, H. J. (2008). FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction. Methods Mol Biol 440: 349-369.

简介

我们开发了内吞标记FM1-43的光转换方案,然后在果蝇神经肌肉接头处进行突触引物的电子显微镜分析。 即使在释放速率非常低时,例如在自发释放模式期间,该方案允许检测染色的突触小泡。 该制剂装载有FM1-43染料,经预先固定,处理和照射,以使染料转变为染料,然后进行常规电子显微镜处理。 该方法能够在电子显微照片下清楚鉴定染色的突触小泡。
【背景】神经元发射体通过突触小泡与神经元质膜的融合而释放。囊泡可以自发融合或响应动作电位。随后,囊泡通过内吞作用获得回收。通过分子生物学,电生理学和显微镜的工具广泛研究了突触小泡回收的分子机制(Slepnev和De Camilli,2000; Sudhof,2004; Rizzoli和Betz,2005; Kavalali,2006)。加载内参标记FM1-43与染料光转换耦合,然后进行电子显微镜分析是一种强大的技术,允许调查和测量回收囊泡池(Harata et al。,2001; Schikorski and Stevens,2001; Rizzoli和Betz, 2004)。果蝇神经肌肉接头(NMJ)是具有明确定义的突触引物的有利制剂,其能够快速产生具有突变突触蛋白的细胞系和严格评估囊泡回收池(Akbergenova和Bykhovskaia,2009; Denker等人,2009)。突触传播领域的一个根本问题是诱发和自发传播是否利用相同的回收池。为了解决这个问题,需要测量在没有刺激的情况下使用的回收池。这是一个具有挑战性的问题,因为自发释放和循环利用率很低。我们已经开发了FM1-43负载协议,随后进行染料光转换和EM分析,可以在果蝇NMJ自发诱发传播过程中对回收池进行严格评估(Sabeva等,2017)。

关键字:电子显微镜检查, 光转换, FM1-43, 果蝇, 突触小泡, 回收池, 神经肌肉接头

材料和试剂

  1. 样品制备
    1. 手套
    2. 长袖实验室外套
    3. 样品瓶带有卡帽,尺寸4毫升(电子显微镜科学,目录号:64250)
    4. Falcon 35毫米培养板(康宁,纽约州)
    5. 黑腹果蝇飞行股票:Canton S(布卢明顿果蝇 Stock Center,目录号:FBst1000081)和 cpx SH1 ( cpx - / - )(Huntwork和Littleton,2007)
    6. 含有75μMAdasep-7(Biotium,目录号:70029)(见注1)的Ca 2 +
    7. FM1-43(Thermo Fisher Scientific,Invitrogen TM,目录号:T35356),10μM
    8. 在HEPES中的100mM NH 4 Cl(Sigma-Aldrich,目录号:A9434)
    9. HEPES中的1.5 mg / ml DAB(Agilent Technologies,DAKO,OEM)
      注意:化合物可用于液体或药片中的片剂。
    10. 使用4%OsO 4储备溶液(Electron Microscopy Sciences,目录号:19150)在90mM二甲胂酸盐缓冲液中制备的1%四氧化锇(OsO 4+)
    11. 升序的丙酮系列50,70和90%由100%
      制备
    12. 在水中制备的2%乙酸双氧铀(Electron Microscopy Sciences,目录号:22400)
    13. 用分子筛脱水剂Fluka(Fluka Analysis,Sigma-Aldrich,目录号:270725)脱水的丙酮100%(Sigma-Aldrich,目录号:270725)
    14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
    15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P3911)
    16. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M9272)
    17. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
    18. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S6014)
    19. 海藻糖(Sigma-Aldrich,目录号:T9531)
    20. 蔗糖(Sigma-Aldrich,目录号:S1888)
    21. HEPES-Na盐(Sigma-Aldrich,目录号:H7006)
    22. HEPES(Sigma-Aldrich,目录号:H3375)
    23. 多聚甲醛(Electron Microscopy Sciences,目录号:15710)
    24. 戊二醛(Electron Microscopy Sciences,目录号:16220)
    25. 二甲胂酸钠缓冲液(Electron Microscopy Sciences,目录号:11653)
    26. HL3果汁生理盐水(见食谱)
    27. 预固定解决方案(请参阅配方和注2)
    28. HEPES缓冲盐水(见食谱)
    29. 固定解决方案(请参阅配方和注2)

  2. 样品嵌入
    1. ACLAR ® 33C嵌入膜(Electron Microscopy Sciences,目录号:50425-10)
    2. 三粒玉米烧杯:塑料,一次性(电子显微镜科学,目录号:60972)
    3. 平底胶囊(电子显微镜科学,目录号:70021)
    4. Epon(Embed-812)(Electron Microscopy Sciences,目录号:14900)
    5. Nadic甲基酐(NMA)(电子显微镜科学,目录号:19000)
    6. 十二碳烯琥珀酸酐特别蒸馏(DDSA)(电子显微镜科学,目录号:13710)
    7. 2,4,6-三(二甲氨基甲基)苯酚(DMP-30)(Electron Microscopy Sciences,目录号:13600)
    8. ERL-4221(电子显微镜科学,目录号:15004)
    9. D.E.R. 736环氧树脂(用于简化与嵌入812结合的渗透)(电子显微镜科学,目录号:13000)
    10. 壬烯基琥珀酸酐改性(NSA)(电子显微镜科学,目录号:19050)
    11. 二甲基氨基乙烷(DMEA)(Electron Microscopy Sciences,目录号:13300)
    12. 嵌入混合A(见配方)
    13. 嵌入混合B(见配方)

设备

  1. 样品制备
    1. Sylgard 184(世界精密仪器公司,萨拉索塔,佛罗里达州)
    2. 精细镊子(世界精密仪器,型号:#2和#5)
    3. 精细剪刀(世界精密仪器,型号:501233)
    4. 昆虫微针(0.1毫米)(精细科学工具,目录号:26002-10)
    5. 解剖立体变焦显微镜(Nikon Stereozoom Microscope,Nikon Corporation)
    6. A.M.P.I. Master 8刺激器(A.M.P.I.,型号:Master-8)
    7. 吸入电极填充HL3
    8. 带荧光复合显微镜,带有针对FM1-43染料定制的长通辐射过滤器的过滤器立方体
    9. 60x浸水物镜(ZEISS,Thornwood,NY)
    10. 水银灯
    11. Biowave(Ted Pella,Redding,CA)
    12. 480±10带通滤波器
    13. PELCO ® R1单速转子在35°位置(Ted Pella,型号:PELCO ® R1)
    14. 通风柜

  2. 样品嵌入
    1. 烤箱粘合剂(Tuttlingen,德国)

  3. 超薄部分准备和图像捕获
    1. 超薄切片机(Leica,型号:Leica EM UC6)
    2. Formvar /碳涂层2 x 1 mm槽铜格栅(电子显微镜科学,目录号:FCF-2010-Cu)
    3. Ultra Diatome金刚石切片刀,湿(电子显微镜科学,目录号:27-US)
    4. UltraTrim干燥室温度刀,4.0mm(电子显微镜科学,目录号:UTT-40-R)
    5. 睫毛带柄(Ted Pella,目录号:119)
    6. 双边不锈钢刀片(电子显微镜科学,哈特菲尔德,PA)
    7. 镊子(电子显微镜科学,哈特菲尔德,PA)
    8. JEOL 100 CX电子显微镜配备浜松数码相机和AMT软件

软件

  1. ImageJ(National Institutes of Health)用于图像分析
  2. 用于图像分析的Adobe Photoshop(Adobe Systems)软件

程序

  1. 在HL3溶液中解除果蝇黑腹果蝇幼虫
    1. 选择第三龄幼虫(L3),并将其置于Sylgard皿中。
    2. 将L3置于前(头/口设备)和后部,背侧。
    3. 将含有4-5毫升HL3溶液(食谱1)的夹心幼虫浸入Sylgard培养皿中。
    4. 纵向剖开背侧,取出内脏(肠,脂肪体和气管)。
    5. 拉伸并固定L3的其余部分。
    6. 可以使用几个肌肉。在这项研究中,实验在肌肉6和7中的Ib boutons进行。(图1A)


      图1.三龄期幼虫解剖。A.肌肉纤维7,6,13,12和V形的高放大图像; B.L3中的腹侧肌肉的图像,其中神经(黑色箭头)被吸入连接到刺激器的吸引电极; C和D.调整加载FM1-43(WT幼虫加载30秒)的预先固定样品的照明时间。 C.DIC图像显示9分钟的照明时间产生成功的光转换。突触引物可以被识别为在肌肉表面几乎看不见的黑斑(白色箭头)。 D. 15分钟的照明时间是过量的,从强烈的DAB沉淀可以看出,在NMJ长度上观察到大的黑点(白色箭头)。比例尺= 10微米。

  2. 突触小泡与FM1-43染料的活性依赖性标记
    注意:在低光条件下执行以下步骤以保护FM1-43染料的荧光团成分。用箔盖住FM1-43染料,或在室温下在黑暗中工作
    1. 用含有10μMFM1-43的HL3替换HL3溶液。
    2. 在没有刺激(自发回收池)的情况下装载,留下装载时间(在我们的研究中使用10,30,120和600秒)的准备。
    3. 对于主动加载,使用头阶段控制器将神经拉入连接到刺激器的抽吸电极。吸收神经的末端使用刺激方案进行低频刺激,5分钟,5 Hz。一旦刺激完成,快速取出FM1-43染料(图1B)。 (Verstreken等人,2008)采用的技术。
    4. 在染料负载时,在含有75μMAdvasep-7的Ca 2+ / +无游离HL3盐水中快速洗涤3次,每次30秒。

  3. 固定术
    1. 在室温下将制剂(配方2)前缀15分钟。
    2. 在HEPES缓冲盐水中洗3次5分钟(食谱3)
    3. 将制备物保留在100mM NH 4 Cl中10分钟以淬灭固定剂的自体荧光。
    4. 用HEPES缓冲液洗涤NH 4 SCl 2溶液2次,5分钟
  4. FM1-43光转换
    1. 在含有1.5mg / ml DAB的HEPES缓冲液中预处理10分钟。
    2. 将样品置于荧光显微镜下。在室温下,确定60倍水浸物镜下的感兴趣区域。
    3. 使用具有最大光强度的480±10带通切除滤光片的汞灯照亮样品8-10分钟。染料会随时间漂白。通过周期性切换到明亮的视野来观察光电转换的过程。随着光转换的发生,深棕色DAB沉淀物定位在照射区域。需要调整照明时间,如图1C和1D所示
  5. 后固定
    1. 在HEPES中洗涤样品三次5分钟。
    2. 将制剂浸入固定剂溶液(配方4)中,并将样品在BioWave中以100W固定2分钟。将温度插入物靠近准备物,以监测温度波动。不允许温度超过25-26°C。
    3. 从Sylgard菜中小心地取出准备。将准备物转移到带有卡帽的样品瓶中。
    4. 将样品在90mM二甲胂酸钠缓冲液(pH 7.4)中洗涤两次,持续5分钟
  6. EM样品处理
    注意:以下步骤(除非另有说明)在室温下进行。
    1. 四氧化锇后固定和乙酸铀酰染色
      1. 在室温下将制剂在1%OsO 4溶液中孵育1小时。未使用的溶液可以冷藏1个月以上。在通风橱下执行所有步骤,使用手套和长袖实验室外套。使用500μl该溶液浸泡样品。 (注4)
      2. 用90mM二甲胂酸盐缓冲液洗涤5分钟
      3. 用去离子水洗涤5分钟
      4. 在2%乙酸铀酰中孵育制剂30-60分钟。未使用的溶液可以冷藏至多2个星期。
      5. 用去离子水洗涤5分钟两次。
    2. 脱水
      1. 制备含有50%,70%,90%和100%丙酮的单独溶液。
      2. 向样品中加入1ml 50%丙酮10分钟
      3. 向样品中加入1ml 70%丙酮10分钟
      4. 向样品中加入1ml 90%丙酮10分钟
      5. 向样品中加入1ml 100%丙酮10分钟
    3. 嵌入
      1. 将2体积的嵌入树脂(注3)与一体积的100%丙酮混合。在连续旋转下加入制剂1 h或过夜。
      2. 将样品保持在树脂中至少18小时(过夜)。
      3. 在立体显微镜下,将准备放在ACLAR电影上,然后识别和修剪感兴趣的区域。
      4. 将准备放在新的ACLAR胶片上,肌肉朝下,角质层朝上。将填充有聚合树脂的嵌入胶囊倒置在样品的顶部。在60℃下聚合24-36小时。
      5. 删除ACLAR电影。这将使样品附着在嵌入胶囊上。
    4. 薄切片和EM成像
      1. 从嵌入胶囊中取出块体,并将块装入超薄切片机的样品架上。
      2. 在立体显微镜下,用剃刀刀片标记感兴趣的区域。标记区域应该是适合插槽格栅的梯形。
      3. 将块修剪在轮廓区域上。
      4. 将样品架插入超薄切片机中,将梯形的长边对准修剪刀边。修剪15-20微米,并移动刀来修剪梯形的第二个长边。修剪15-20微米。对梯形的短边重复相同(图2和视频1-5)。
      5. 将块面对准与金刚石切割刀的边缘完全平行。
      6. 切割薄片(50-60 nm)
      7. 收集单个部分(或串联收集部分),并将它们放在2 x 1 mm插槽网格上。
      8. 在拍摄图像之前让部分干燥。
      9. 使用常规TEM图像显示。


        图2.样品块精细修剪。 在固定和平面嵌入式果蝇幼虫中,感兴趣的区域,6和7肌肉用剃刀刀片概述。在用剃刀刀片进行粗糙的修整和消除树脂时,块的面部用修剪刀成形。 A.微调。刀的边缘与梯形顶部对齐。 B.垂直定位的梯形,修剪刀的左边缘与梯形的顶部对齐。 C.具有平行顶部和底部以及稍微倾斜的侧面的细微梯形。
        注意:平行的顶部和底部对于在串行切片期间形成色带很重要。

        Video 1. Rough trapezoid trimming

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        Video 2. Fine trapezoid trimming, part one. Trimming trapezoid top.

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        Video 3. Fine trapezoid trimming, part 2. Once sectioned the trapezoid top the trimming knife is retracted and positioned to trapezoid bottom without additional alignment.

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        Video 4. Fine trapezoid trimming, part 3. Trimming trapezoid bottom.

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        Video 5. Serial sectioning

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数据分析

  1. 使用光转换程序,我们调查了缺乏突触前蛋白复合物的果蝇幼虫中的回收囊泡池( cpx / sup>)(Huntwork和图3A),其能够提高自发释放速率。相比之下,在自发加载期间在WT boutons中观察到的回收池可以忽略不计(数据未显示,参见[Sabeva等人,2017])。在神经刺激期间装载的 cpx 中,与WT制剂相比,回收池也增加(图3A和3B)表明单独的回收池用于自发和诱发释放模式(Sabeva等人,2017)。


    图3.显示回收囊泡池的代表性照片。 A. 在缺席的情况下加载boutons刺激10,30或120秒。在5Hz的神经刺激期间加载5分钟的WT和 cpx 。光转化的标记囊泡(黑色),未标记的囊泡(灰色)。比例尺= 500nm。

  2. 详细的数据处理分析和复制,包括应用的统计测试可以在原始手稿中找到,见Sabeva等人,2017年。

笔记

  1. Advasep-7是β-环糊精衍生物。它可以作为染料清除剂,减少背景荧光
  2. 16%多聚甲醛溶液和25%戊二醛溶液可购买于10ml安瓿。
  3. 嵌入树脂:通过混合等体积的嵌入混合物A(配方5)和嵌入混合物B(配方6),在新的一次性250ml烧杯中制备。使用木制舌头按摩器彻底混合。在真空干燥器中脱气(消除气泡)5分钟,或在室温下放置30分钟。在室温下密封保存,并在同一天使用,或等分并储存于-20°C
  4. EM样品处理过程中的所有步骤均在振动筛或样品旋转器中进行

食谱

  1. HL3 生理盐水溶液


  2. 预固溶液


  3. HEPES缓冲盐水


  4. 固定解决方案


  5. 嵌入混合A
    混合准备:
    20 g EMBed-812
    11 g DDSA
    9克NMA
    0.8 ml DMP-30
    在250毫升Tri-Corn一次性烧杯中 使用木制舌头按压器彻底混合
  6. 嵌入混合B
    混合准备:
    10克ERL
    6克DER
    26克NSA
    0.4毫升DMAE
    在250毫升Tri-Corn一次性烧杯中 使用木制舌头按压器彻底混合

致谢

这项工作得到了国家卫生研究院授予的R01 MH099557和NIH NINDS SNRP Grant 5U54NS083924的支持。

参考

  1. Akbergenova,Y。和Bykhovskaia,M。(2009)。增强内体内吞途径增加量化大小。分子细胞Neurosci 40(2):199-206。
  2. Denker,A.,Krohnert,K.和Rizzoli,SO(2009)。  在果蝇神经肌肉接头中重新检查突触小泡池的定位。 J Physiol 587(Pt 12):2919-2926。
  3. Harata,N.,Ryan,TA,Smith,SJ,Buchanan,J.和Tsien,RW(2001)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih通过FM 1-43光转换可视化回收海马神经元中的突触小泡。 98(22):12748- 12753.
  4. Huntwork,S.and Littleton,JT(2007)。  复合蛋白融合钳调节自发性神经递质释放和突触生长。 Nat Neurosci 10(10):1235-1237。
  5. Kavalali,ET(2006)。  突触小泡再利用及其影响。神经科学家 12(1):57-66。
  6. Rizzoli,SO和Betz,WJ(2004)。  易于释放的突触小泡池的结构组织。 科学 303(5666):2037-2039。
  7. Rizzoli,SO和Betz,WJ(2005)。  Synaptic囊泡池。 Nat Rev Neurosci 6(1):57-69。
  8. Sabeva,N.,Cho,W.R.,Vasin,A.,Gonzalez,A.,Littleton,T.J。和Bykhovskaia,Maria。 (2017)。复合蛋白突变体显示驱动诱发和自发神经传递的回收途径之间的部分分离。 Neurosci 37:383-396。
  9. Schikorski,T。和Stevens,CF(2001)。  功能定义的突触小泡种群的形态相关性。 Nat Neurosci 4(4):391-395。
  10. Slepnev,VI和De Camilli,P。(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/11257904"target ="_ blank" >网格蛋白依赖性突触小泡内吞的附属因素。 Nat Rev Neurosci 1(3):161-172。
  11. Sudhof,TC(2004)。突触小泡循环。 Annu Rev Neurosci 27:509-547。
  12. Verstreken,P.,Ohyama,T.和Bellen,H.J。(2008)。 FM 1-43在上标记突触小泡池果蝇神经肌肉接头。方法Mol Biol 440:349-369。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sabeva, N. and Bykhovskaia, M. (2017). FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction. Bio-protocol 7(17): e2523. DOI: 10.21769/BioProtoc.2523.
  2. Sabeva, N., Cho, W. R., Vasin, A., Gonzalez, A., Littleton, T. J. and Bykhovskaia, Maria. (2017). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci 37: 383-396.
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