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May 2020

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Preparation of Synaptoneurosomes to Study the Synapse in the Murine Cerebral Cortex
用于研究小鼠大脑皮层突触的突触神经小体的制备   

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Abstract

The synapse is a complex structure where the transmission of information takes place. Synaptic dysfunction is one of the earliest pathophysiological events in several diseases, such as traumatic brain injury, cerebral ischemia, and neurodegenerative diseases. Thus, a methodology to study synaptic structure and function is crucial for the development of potential strategies for the treatment of many neurological diseases. Synaptoneurosomes (SNs) are structures assembled by the sealed presynaptic bouton and the attached post-synaptic density. Despite the fact that for a long time it has been recognized that SNs are a powerful tool to study synaptic function, composition, and structure, its use has been limited by the requirement of relatively large amounts of material to successfully isolate them. Here we describe a three-step centrifugation procedure performed under hypotonic conditions to isolate SNs from small volumes of the cerebral cortex.


Graphic abstract:



Schematic flowchart for the preparation of synaptoneurosomes.

Keywords: Synapse (突触), Synaptoneurosomes (突触体), Subcellular fractionation (次细胞分离法), Brain (大脑), Synaptic compartment (突触腔), Tripartite synapse (三方突触)

Background

The synapse is a structure assembled by a presynaptic bouton attached to a postsynaptic terminal and ensheathed by an astrocytic elongation (Halassa et al., 2007). The last decade has witnessed the development of a large number of highly sophisticated techniques to study its structure and function. However, SNs are still used by a large number of investigators because they are relatively easy to isolate and yield valuable information on synaptic structure and function. The preparation of SNs is a modified cell-fractioning procedure that is performed under specific hypotonic conditions, and allows the isolation of the presynaptic boutons and attached postsynaptic elements. In the initial phases of their identification, they were called cell-free responsive preparations because their response to hormones (Chasin et al., 1974; Horn and Phillipson, 1976). This was followed years later by a detailed description of a procedure to isolate a suspension of snowman-shaped structures (synaptoneurosomes) assembled by a sealed presynaptic terminal (synaptosomes) attached to a postsynaptic sac (neurosomes; Hollingsworth et al., 1985). Since then several modifications have been introduced to their preparation, including the use of different hypotonic solutions and rotors (Rao and Steward, 1991; Villasana et al., 2006). However, despite their importance, the use of SNs has been limited by the requirement of relatively large amounts of tissue for their preparation. To address this limitation, here we describe a modified procedure to isolate SNs from small amounts of fresh and frozen brain tissue, and cultured neurons (Diaz et al., 2017 and 2020). This protocol has been used to successfully study the tripartite synapse in neuronal-astrocytic co-cultures (Diaz et al., 2019).

Materials and Reagents

  1. 15 ml Conical Centrifuge tubes (any brand)

  2. 10 ml, Open-Top Thickwall Polycarbonate tube, 16 x 76 mm (Beckman Coulter, catalog number: 355630 )

  3. 2.2 ml, Open-Top Thinwall Ultra-Clear tube, 11 x 34 mm (Beckman Coulter, catalog number: 347356 )

  4. Pipette tips

  5. 60 mm cell culture dish (EMD Millipore, catalog number: CLS430589 )

  6. Nitrocellulose Membrane (Bio-Rad, catalog number: 1620112 )

  7. One 8- to 12-week-old mouse

  8. Isoflurane, USP (Piramidal Critical Care, catalog number: 66794-013-25 )

  9. Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, catalog number: 46430 )

  10. Protease inhibitors, complete tablets EDTA-free EASYpack (Roche, catalog number: 04693132001 )

  11. Phosphatase inhibitors, PhosSTOP EASYpack (Roche, catalog number: 04906837001 )

  12. Rabbit anti-Syntaxin 1 antibody (1:1,000; EMD Millipore, catalog number: AB5820-50UL )

  13. Mouse anti-PSD-95 antibody 6G6-1C9 (1:1,000; Novus Biologicals, catalog number: NB300-556 )

  14. Mouse anti-β-Actin antibody (1:100,000; Sigma-Aldrich, catalog number: A1978 )

  15. Mouse anti-Synaptophysin antibody (1:1,000; EMD Millipore, catalog number: MAB5258 )

  16. Rabbit anti-Histone H3 antibody (1:1,000; Cell signaling, catalog number: 4499 )

  17. IRDye 800CW Donkey anti-Rabbit IgG secondary antibody (1:10,000; LI-COR, catalog number: 926-32213 )

  18. IRDye 680RD Donkey anti-Mouse IgG secondary antibody (1:10,000; LI-COR, catalog number: 926-68072 )

  19. Intercept (TBS) Blocking Buffer (LI-COR, catalog number: 927-60001 )

  20. Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad, catalog number: 4568123 )

  21. Mouse Surgical Kit (Kent Scientific, INSMOUSEKIT)

  22. Sucrose (Fisher Chemical, catalog number: S612 )

  23. Trizma base (Sigma-Aldrich, catalog number: T6066-5KG )

  24. RIPA buffer (TEKnova, catalog number: R3792 )

  25. Phosphate Buffered Saline 10x (Growcells, catalog number: RGF-6235 )

  26. Regular ice

  27. Ficoll (GE Healthcare, catalog number: 17-0300-10 )

  28. 100 mM EGTA (see Recipes)

  29. 10 mM Tris pH 8.1 (see Recipes)

  30. Homogenization buffer (HB) (see Recipes)

  31. Ficoll solutions (see Recipes)

  32. Discontinuous Ficoll gradient (see Recipes)

  33. Tris-buffer Saline (TBS) 20x (see Recipes)

  34. TBS-Tween (TBS-T) (see Recipes)

Equipment

  1. Micropipettes set

  2. -80 °C freezer

  3. Swinging-bucket clinical centrifuge (Beckman Coulter) or any centrifuge with temperature control compatible with 15 ml tubes

  4. Optima Max-XP ultracentrifuge (Beckman Coulter)

  5. TLS-55 Swinging-Bucket Rotor (Beckman Coulter)

  6. MLS-50 Swinging-Bucket Rotor (Beckman Coulter)

  7. Universal laboratory stirrer IKA EuroStar 60 Digital (Sigma-Aldrich, catalog number: Z766976 )

  8. Potter-Elvehjem Tissue Homogenizer 5 ml (Omni International, catalog number: 07-358034 )

  9. Mini-PROTEAN Tetra Cell for Ready Gel Precast Gels (Bio-Rad, catalog number: 1658005EDU )

  10. Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, catalog number: 1703940 )

  11. Odyssey Fc Imaging System (Li-COR)

Software

  1. ImageStudio v5.2 (Li-COR)

Procedure

  1. SN from brain tissue (work on ice all the time)

    1. Induce anesthesia in the mouse using the isoflurane.

    2. Perfuse the mouse intracardially with cold PBS 1x.

    3. Open the skull, remove the meninges, and dissect the brain cortex (or the anatomical area of interest) in a 60 mm cell culture dish. 40-100 mg of brain tissue is enough for one experiment.

    4. Save the other hemisphere (see Step A8).

    5. Chop the tissue using small scissors in 1 ml of HB.

    6. Homogenize tissue (homogenate 1, H1) in 4 ml of HB using a Potter-Elvehjem glass/teflon homogenizer.

    7. Stroke 8 times at 800 rpm on ice.

    8. Homogenize the other hemisphere with 1 ml of RIPA buffer containing protease and phosphatase inhibitors, centrifuge at 18,000 x g at 4 °C, and recover supernatant with a pipette and store at -80 °C. This will be used to prepare whole brain extract (WBE) which will constitute an internal control for SNs enrichment (see Data analysis).


  2. Centrifugation

    1. Centrifuge homogenate H1 at 2,000 x g for 5 min at 4 °C in a swinging-bucket clinical centrifuge.

    2. Transfer the supernatant (S1) to a 10 ml, Open-Top Thickwall Polycarbonate tube, 16 x 76 mm, discard pellet (P1), and centrifuge the S1 in an MLA-55 fixed angle rotor at 30,000 x g for 10 min at 4 °C to obtain pellet 2 (P2; this pellet does not disrupt easily because is dense and thick), discard the supernatant 2 (S2) with a pipette (work on ice).

    3. Resuspend P2 in 400 µl of HB (homogenate 2; H2) by pipetting several times (work on ice).


  3. Discontinuous Ficoll gradient

    1. Layer the H2 on top of a 5-10.3% discontinuous Ficoll gradient (Recipe 4) and centrifuge at 45,000 x g for 20 min at 4 °C in a TLS-55 rotor using a Beckman Optima Max-XP tabletop ultracentrifuge.

    2. Collect 400 μl of the white layer in the 5-10.3% Ficoll interface that contains SNs.

      (Figure 1; this fraction may also be used for other procedures or analyses, such as isolation of presynaptic terminals, synaptic vesicles and postsynaptic terminals, as well flow cytometry, omics, and electron microscopy).



      Figure 1. 2.2 ml, Open-Top Thinwall Ultra-Clear tube at the end of Step C1. Note the white 5%/10.3% interface band containing SNs that will be collected in step C2. The pellet contains mainly mitochondria and cell debris.


    3. Complete volume with 1,400 μl of HB.

    4. Centrifuge the SNs layer at 30,000 x g for 10 min at 4 °C in a TLS-55 rotor using a Beckman Optima Max-XP tabletop ultracentrifuge. Then, using a 200 μl tip pipette connected to the vacuum remove the supernatant (the SNs pellet does not disrupt easily because is dense and thick).

    5. Resuspend the SNs pellet using 150 μl of RIPA buffer containing protease and phosphatase inhibitors store at -80 °C.

Data analysis

  1. Run an SDS-PAGE

    1. Quantify proteins using the laboratory routine protocol.

    2. Mount the precast gel and load 10 μg of SNs, WBE (internal control), and a molecular weight marker.

    3. Run gel.


  2. Immunoblot and interpretation

    1. Transfer proteins to a nitrocellulose membrane.

    2. Block membrane with blocking buffer.

    3. Cut membrane between the 55 and 72 kDa molecular weight marker.

    4. Incubate the upper half with anti-PSD-95 antibody.

    5. Incubate the lower half with antibodies against syntaxin 1 and synaptophysin.

    6. Incubate overnight at 4 °C with gentle agitation.

    7. Wash membranes using TBS-T and incubate them with specific LI-COR secondary antibodies mix for 1 h at room temperature.

    8. Wash membranes with TBS-T and detect signal using the Odyssey Fc Imaging System.

    9. Synaptophysin is a marker of pre-synaptic vesicles (Kwon and Chapman, 2011); Syntaxin 1 detects the presynaptic membrane (Bennett et al., 1992), and PSD-95 is a scaffold protein of the postsynaptic compartment (Hunt et al., 1996). A successful procedure should enrich the pre- and post-synaptic markers in the SNs fraction as compared to WBE (Figure 2).



      Figure 2. Representative Western blot analysis. Whole brain extracts (WBE) and synaptoneurosomes (SNs) prepared from the cerebral cortex of a C57 mouse and immunoblotted with antibodies against syntaxin 1, synaptophysin, PSD-95, and histone H3.


    10. Remove antibodies signal from the membranes using Restore PLUS Western Blot Stripping Buffer 20 min at room temperature.

    11. Wash membrane with TBS-T.

    12. Incubate overnight the lower half of the membrane at 4 °C and gentle agitation with antibodies against H3 and β-Actin.

    13. Wash membranes using TBS-T and incubate them with specific LI-COR secondary antibodies mix for 1 hour at room temperature.

    14. Detect signal using the Odyssey Fc Imaging System.

    15. H3 is a nuclear marker; β-Actin is a loading control. SNs should be immunoreactive to β-Actin but not H3 (Figure 2).

Notes

  1. This protocol requires basic knowledge of laboratory and animal surgical procedures.

  2. We recommend to work on ice and/or 4 °C for each step.

  3. For this protocol, we used an 8-week old C57bl/6J male mouse. However, we have also used it to isolate synaptoneurosomes from 3- days/ and 12 months/old male and female mice. To our knowledge, the strain and genetic background are not limitations for this procedure.

  4. 40-100 mg of tissue is enough for one experimental point, however, this amount could be further reduced by using smaller tubes and/or changing the volumes and/or concentrations of the Ficoll gradient.

  5. In our experience, WBE and SNs can be stored for at least 1 year at -80 °C. We recommend to use small aliquots to avoid freeze-thaw cycles.

  6. In our experience, following their preparation the solutions described below can be stored for at least 1 year.

Recipes

  1. 100 mM EGTA

    1. Dissolve 3.8 g of EGTA in 100ml of 10 mM Tris pH 8.1

    2. Store solution at room temperature

  2. 10 mM Tris, pH 8.1

    1. Dissolve 2.42 g of Trizma base in 1.8 L of MilliQ grade H2O

    2. Adjust pH 8.1 with HCl/NaOH as needed

    3. Add water to obtain 2 L final volume

    4. Store at room temperature

  3. HB (0.25 M Sucrose/0.5 mM EGTA buffer in 10 mM Tris, pH 8.1)

    1. For 1 L: 85.57 g Sucrose, 5 ml of 100 mM EGTA in 10 mM Tris pH 8.1, store at 4 °C

    2. Add protease and phosphatase inhibitors to 50 ml of HB

  4. Ficoll solutions

    1. 5% Ficoll

      Dissolve 5 g of Ficoll in 100 ml of HB

      Filter using 0.22 μm pore, store at 4 °C

    2. 14.5% Ficoll

      Dissolve 14.5 g of Ficoll in 100 ml of HB

      Filter using 0.22 μm pore, store at 4 °C

    3. 10.3% Ficoll

      Mix 865 μl of Ficoll 14.5% and 345 μl of HB (for two samples), prepare the same day

  5. Discontinuous Ficoll gradient

    Note: Prepare the gradient before start the experiment or during Step B2.

    1. Add 500 μl of 10.3% Ficoll solution into an Open-Top Thinwall Ultra-Clear tubes (11 x 34 mm)

    2. Using the smoothest 1,000 μl pipette available or a 200 μl pipette, add slowly 1,300 μl of 5% Ficoll solution (a 5%/10.3% interface should be visible, if not, discard the gradient and start again using a clean tube)

  6. TBS 20x for 2 L

    1. Dissolve 96 g of Trizma base and 352 g of NaCl in 1 L of Ficoll 5%: dissolve 5 g of MilliQ grade H2O

    2. Adjust pH 7.6 with HCl/NaOH as needed

    3. Add water to obtain 2 L final volume

  7. Tris-buffer Saline Tween 1x

    1. Dilute TBS 20x to 1x with MilliQ grade H2O

    2. Add tween 20 to obtain 0.1% final concentration

Acknowledgments

This work was supported in part by National Institutes of Health Grant NS-NS091201 (to M.Y.), Veterans Administration Merit Award IO1BX003441 (to M.Y.), and American Heart Association Post-Doctoral Fellowship Grant 19POST34380009 (to A.D.).

Competing interests

The authors declare no competing financial interests.

Ethics

Experiments were approved by the Institutional Animal Care and Use Committee (Protocol ID PROTO201700559 from 1/28/2020 to 1/27/2023) of Emory University, Atlanta, GA, USA.

References

  1. Bennett, M. K., Calakos, N. and Scheller, R. H. (1992). Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257(5067): 255-259.
  2. Chasin, M., Mamrak, F. and Samaniego, S. G. (1974). Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain. J Neurochem 22(6): 1031-1038.
  3. Diaz, A., Merino, P., Manrique, L. G., Ospina, J. P., Cheng, L., Wu, F., Jeanneret, V. and Yepes, M. (2017). A Cross Talk between Neuronal Urokinase-type Plasminogen Activator(uPA) and Astrocytic uPA Receptor(uPAR) Promotes Astrocytic Activation and Synaptic Recovery in the Ischemic Brain. J Neurosci 37(43): 10310-10322.
  4. Diaz, A., Merino, P., Manrique, L. G., Cheng, L. and Yepes, M. (2019). Urokinase-type plasminogen activator(uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes. J Cereb Blood Flow Metab 39(11): 2157-2171.
  5. Diaz, A., Merino, P., Guo, J. D., Yepes, M. A., McCann, P., Katta, T., Tong, E. M., Torre, E., Rangaraju, S. and Yepes, M. (2020). Urokinase-type plasminogen activator protects cerebral cortical neurons from soluble Aβ-induced synaptic damage. J Neurosci 40(21): 4251-4263.
  6. Halassa, M. M., Fellin, T. and Haydon, P. G. (2007). The tripartite synapse: roles for gliotransmission in health and disease. Trends Mol Med 13(2): 54-63.
  7. Hollingsworth, E. B., McNeal, E. T., Burton, J. L., Williams, R. J., Daly, J. W. and Creveling, C. R. (1985). Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes. J Neurosci 5(8): 2240-2253.
  8. Horn, A. S. and Phillipson, O. T. (1976). A noradren aline sensitive adenylate cyclase in the rat limbic forebrain: preparation, properties and the effects of agonists, adrenolytics and neuroleptic drugs. Eur J Pharmacol 37(1): 1-11.
  9. Hunt, C. A., Schenker, L. J. and Kennedy, M. B. (1996). PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses. J Neurosci 16(4): 1380-1388.
  10. Kwon, S. E. and Chapman, E. R. (2011). Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons. Neuron 70(5): 847-854.
  11. Rao, A. and Steward, O. (1991). Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes. J Neurosci 11(9): 2881-2895.
  12. Villasana, L. E., Klann, E. and Tejada-Simon, M. V. (2006). Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus. J Neurosci Methods 158(1): 30-36.

简介

[摘要]突触是一个复杂的结构,在此结构中发生信息的传递。小号ynaptic功能障碍是几种疾病,如脑外伤,脑缺血最早的病理生理活动之一,和神经退行性疾病。因此,研究突触结构和功能的方法学对于开发治疗许多神经系统疾病的潜在策略至关重要。突触神经小体(SN)是由密封的突触前钮扣和附加的突触后密度组装而成的结构。尽管很长一段时间以来,人们已经认识到SN是研究突触功能,组成的强大工具,在结构和结构上,其使用受到成功地隔离它们所需的相对大量材料的限制。在这里,我们描述了在低渗条件下执行的三步离心程序,以从少量大脑皮层中分离出SN。


图形摘要:


突触神经体的制备流程图。


[背景]突触是由连接至突触后终端和突触前布顿组装的结构所包围由星形细胞伸长率(Halassa等人,2007 )。在过去的十年中,见证了许多用于研究其结构和功能的高度复杂的技术的发展。但是,SN仍被大量研究者所使用,因为它们相对容易分离并产生有关突触结构和功能的有价值的信息。SNs的制备是在特定的低渗条件下进行的改良的细胞分离程序,可分离突触前突突和附着的突触后元件。在鉴定的最初阶段,它们被称为无细胞响应制剂,因为它们对激素具有响应(Chasin等,197 4 ;Horn和Phillipson ,1976 )。这是年后,随后的过程的详细描述,以分离的悬浮液雪人形结构(的synaptoneurosomes附着于突触后囊由密封的突触前末梢组装)(突触体)(neurosomes ;霍林斯沃思。等人,1985)。从那时起,已经对其制备进行了一些修改,包括使用不同的低渗溶液和转子(Rao和Steward ,1991;Villasana等,2006)。然而,尽管它们很重要,但是由于制备它们需要相对大量的组织,因此限制了SN的使用。为了解决这个限制,在这里我们描述了一种改良的程序,可以从少量新鲜和冷冻的脑组织以及培养的神经元中分离出SN(Diaz等人,2017和2020)。此协议已被成功地用于研究三方突触在神经元-星形细胞共同培养(迪亚兹等人,2019)。

关键字:突触, 突触体, 次细胞分离法, 大脑, 突触腔, 三方突触

材料和试剂
15 ml锥形离心管(任何品牌)
10 ml,开顶厚壁聚碳酸酯管,16 x 76 mm (Beckman Coulter,目录号:355630 )
2.2毫升,敞口薄壁超清管,11 x 34毫米(贝克曼库尔特(Beckman Coulter),目录号:347356 )
移液器技巧
60 mm细胞培养皿(EMD密理博,目录号:CLS430589)
硝酸纤维素膜(Bio- R ad,目录号:1620112)
一只8至12周大的老鼠
异氟烷,美国药典(Piramidal重症护理,目录号:66794-013-25)
Restore PLUS Western Blot剥离缓冲液(Thermo Fisher Scientific,目录号:46430)
蛋白酶抑制剂,完整片剂,不含EDTA EASYpack (Roche,目录号:04693132001)
磷酸酶抑制剂PhosSTOP EASYpack (Roche,目录号:04906837001)
兔抗Syntaxin 1抗体(1:1,000; EMD Millipore,目录号:AB5820-50UL)
小鼠抗PSD-95抗体6G6-1C9(1:1,000; Novus Biologicals,目录号:NB300-556)
小鼠抗β-Actin抗体(1:100,000; Sigma-Aldrich,目录号:A1978)
小鼠抗突触素抗体(1:1,000; EMD Millipore,目录号:MAB5258)
兔抗组蛋白H3抗体(1:1,000;细胞信号传导,目录号:4499)
IRDye 800CW驴抗兔IgG二抗(1:10,000; LI-COR,目录号:926-32213)
IRDye 680RD驴抗小鼠IgG二抗(1:10,000; LI-COR,目录号:926-68072)
拦截(TBS)阻塞缓冲区(LI-COR,目录号:927-60001)
Mini-PROTEAN TGX无污染凝胶(Bio- R ad,目录号:4568123)
鼠标手术套件(Kent Scientific,INSMOUSEKIT)
蔗糖(Fisher Chemical,目录号:S612)
Trizma底座(Sigma-Aldrich,目录号:T6066-5KG)
RIPA缓冲器(TEKnova ,目录号:R3792)
磷酸盐缓冲盐水10x(Growcells ,目录号:RGF-6235)
常规冰
Ficoll (GE Healthcare,目录号:17-0300-10)
100 mM EGTA(请参阅食谱)
10 mM Tris pH 8.1(请参阅食谱)
均质缓冲液(HB)(请参见配方)
Ficoll解决方案(请参阅食谱)
非连续Ficoll梯度(请参见食谱)
Tris-Buffer Saline(TBS)20 x (请参阅食谱)
TBS补间(TBS-T)(请参阅食谱)

设备


微量移液器套装
-80°C冷冻室
摇摆式临床离心机(Beckman Coulter)或任何温度控制与15 ml管兼容的离心机
Optima Max-XP超速离心机(贝克曼库尔特)
TLS-55摆桶式转子(贝克曼库尔特)
MLS-50旋斗式转子(贝克曼库尔特)
通用实验室搅拌器IKA EuroStar 60 Digital(Sigma - A ldrich,目录号:Z766976)
Potter- Elvehjem组织匀浆器5毫升(欧姆尼国际公司,目录号:07-358034)
用于预制凝胶预制凝胶的Mini-PROTEAN Tetra Cell(Bio- R ad,目录号:1658005EDU)
Trans-Blot SD半干电泳转移池(Bio- R ad,目录号:1703940)
奥德赛Fc成像系统(Li-COR)


软件


ImageStudio v5.2 (Li-COR)

程序


来自脑组织的SN(始终在冰上工作)
使用异氟烷在小鼠中诱导麻醉。
心内灌注小鼠冷PBS 1x。
打开头骨,取出脑膜,然后在60毫米细胞培养皿中解剖大脑皮层(或感兴趣的解剖区域)。40-100毫克的脑组织足以完成一项实验。
保存另一个半球(请参阅步骤A8)。
用小剪刀在1毫升HB中切碎组织。
匀浆组织在4ml HB的使用Potter-(匀浆1,H1)Elvehjem玻璃/特氟隆均化器。
在冰上以800 rpm的速度冲击8次。
用1 ml含有蛋白酶和磷酸酶抑制剂的RIPA缓冲液匀浆另一个半球,在4°C下以18,000 xg离心,并用移液管回收上清液并保存在-80°C下。这将被用于制备全脑提取物(WBE),其将构成的SN富集的内部对照(见d ATA分析)。

离心分离
在摇摆式临床离心机中于4°C以2,000 xg的速度将H1匀浆5分钟。
将上清液(S1)转移到10 ml开顶厚壁聚碳酸酯管(16 x 76 mm)中,将卡片沉淀(P1),然后将S1在MLA-55固定角转子中以30,000 xg的速度离心4分钟10分钟°C以获得沉淀2(P2;该沉淀不容易破裂,因为它稠密且稠密),用移液管弃去上清液2(S2)(在冰上工作)。
通过移液几次(在冰上工作)将P2重悬于400 µl HB(匀浆2; H2)中。

非连续Ficoll梯度
层中的H 2在一个5的顶部- 10.3%的不连续的Ficoll梯度(配方4)和离心机中以45000 ×g下在用Beckman的Optima最大-XP桌面超速离心机一个TLS-55转子20分钟在4℃下。
收集400 μ升在5-10.3%白色层的聚蔗糖包含的SN接口。
(图1;该部分也可用于其他操作或分析,例如分离突触前末端,突触小泡和突触后末端,以及流式细胞仪,组学和电子显微镜检查)。






图1.步骤C1结束时的2.2 ml开顶薄壁超透明管。请注意,包含SN的白色5%/ 10.3%白色界面带将在步骤C2中收集。沉淀物主要包含线粒体和细胞碎片。


1,400个完整卷 μ升HB的。
离心30,0所述SNS层00 ×g离心10分钟,在TLS-55转子用Beckman的Optima最大-XP桌面超速离心机在4℃下。然后,使用一个200 μ升连接到真空吸管尖端除去上清液(颗粒不容易破坏,因为是致密的和厚的SNS) 。
重悬使用150所述SNS沉淀μ升RIPA缓冲液含有蛋白酶和磷酸酶抑制剂存储在-80℃。

数据分析


运行SDS-PAGE
使用实验室常规规程定量蛋白质。
安装预制凝胶和负载10 μ克SNS,WBE(内部对照)的,和分子量标记。
运行凝胶。

免疫印迹和解释
将蛋白质转移到硝酸纤维素膜上。
用封闭缓冲液封闭膜。
在55至72 kDa分子量标记之间切膜。
用抗PSD-95抗体孵育上半部分。
用抗语法1和突触素的抗体孵育下半部分。
在4°C轻轻搅拌下孵育过夜。
使用TBS-T,并与特定的孵育他们洗膜LI-COR的第二抗体混合对于在室温下1个小时。
用TBS-T清洗膜并使用Odyssey Fc成像系统检测信号。
突触素是突触前囊泡的标志物(Kwon和Chapman ,2011 );Syntaxin 1检测突触前膜(Bennett等,1992 ),而PSD-95是突触后区室的支架蛋白(Hunt等,1996 )。与WBE相比,成功的手术应在SNs分数中丰富突触前和突触后标记(图2)。





图2 。代表性的蛋白质印迹分析。全脑提取物(WBE)和synaptoneurosomes从C57小鼠的大脑皮层制得并用抗抗体进行免疫印迹(SNS)突触1,突触素,PSD-95 ,和组蛋白H3。


在室温下使用Restore PLUS Western Blot Stripping Buffer 20分钟,从膜上去除抗体信号。
用TBS-T清洗膜。
将膜下半部分在4°C下孵育过夜,并用抗H3和β-肌动蛋白的抗体轻轻搅动。
使用TBS-T,并与特定的孵育他们洗膜LI-COR的第二抗体混合对于在室温下1小时。
使用Odyssey Fc成像系统检测信号。
H3是核标记;β-肌动蛋白是一个加载控件。SN应该对β-Actin具有免疫反应性,但对H3不具有免疫反应性(图2)。

笔记


该协议需要实验室和动物手术程序的基础知识。
我们建议每一步在冰和/或4°C下工作。
对于此协议,我们使用了8周大的C57bl / 6J雄性小鼠。但是,我们还用它来分离了3天和12个月大的雄性和雌性小鼠的突触神经小体。据我们所知,菌株和遗传背景不是该方法的限制。
对于一个实验点,40-100 mg的组织就足够了,但是,可以通过使用较小的试管和/或更改Ficoll梯度的体积和/或浓度来进一步减少该量。
根据我们的经验,WBE和SNS可以被存储为在-80℃下至少1年。我们建议使用小等分试样以避免冻融循环。
根据我们的经验,在准备好以下解决方案后,它们可以保存至少一年。 

菜谱


100毫米EGTA
将3.8 g EGTA溶于100 ml 10 mM Tris pH 8.1中
将溶液储存在室温下
10 mM Tris,pH 8.1
溶解2.42克的的Trizma碱在1.8升的的MilliQ等级H 2 ö
根据需要用HCl / NaOH调节pH值8.1
加水获得2升最终体积
室温保存
HB(0.25 m蔗糖/0.5 mM EGTA缓冲液,溶于10 mM Tris中,pH 8.1)
对于1 L:85.57 g蔗糖,10 ml Tris pH 8.1中的5 ml 100 mM EGTA,储存在4°C
将蛋白酶和磷酸酶抑制剂添加到50 ml HB中
Ficoll解决方案
5%菲科尔
D将5 g Ficoll溶于100 ml HB


使用过滤器0.22 μ米孔径,储存在4℃下


14.5%菲科尔
D将14.5 g Ficoll溶于100 ml HB


使用0.22滤波器μ米孔径,储存在4℃下


10.3%菲科尔
中号IX 865 μ升的聚蔗糖14.5%和345 μ升HB的(对于两个样品),准备在同一天


非连续Ficoll梯度
Ñ OTE :P repare梯度之前开始实验或在小号TEP B2 。


加入500 μ升10.3%聚蔗糖溶液成开顶薄壁超清晰管(11 x 34英寸MM)
使用最平滑的1 ,000 μ升可用吸管或200 μ升移液管,缓慢加入1300 μ升的5%聚蔗糖溶液(5%/ 10.3%接口应该是可见的,如果不是,则丢弃该梯度,并使用一个重新开始清洁管)
TBS 20x 2升
将96 g Trizma碱和352 g NaCl溶于1 L的Ficoll 5%:溶解5 g的M illi Q级H 2 O
根据需要用HCl / NaOH调节pH 7.6
加水获得2升最终体积
Tris缓冲盐水吐温1 x
稀TBS 20X 1倍与中号ILLI Q等级H 2 ö
加入吐温20以获得0.1%的最终浓度

致谢


这项工作得到了美国国立卫生研究院补助金NS-NS091201,退伍军人管理局优秀奖IO1BX003441(补助金)和美国心脏协会博士后奖学金19POST34380009(补助金)的部分支持。


利益争夺


作者宣称没有任何竞争性的经济利益。


伦理


实验得到美国佐治亚州亚特兰大市埃默里大学的机构动物护理和使用委员会(协议编号PROTO201700559,日期为1/28/2020至1/27/2023)批准。


参考文献


              Bennett,MK,Calakos ,N。和Scheller ,RH(1992)。Syntaxin:一种突触蛋白,与突触小泡对接在突触前活性区有关。科学257(5067):255-259。
              Chasin ,M.,Mamrak ,F.和Samaniego,SG(1974)。豚鼠脑中无细胞激素响应腺苷酸环化酶的制备及其性质。Ĵ神经化学杂志22(6):1031至1038年。
Diaz,A.,Merino,P.,Manrique,LG,Ospina,JP,Cheng,L.,Wu,F.,Jeanneret ,V.和Yepes ,M.(2017)。神经元尿激酶型纤溶酶原激活物(uPA)与星形胶质uPA受体(uPAR)之间的相互促进在缺血性脑中促进星形胶质细胞活化和突触恢复。Ĵ神经科学37(43):10310-10322。
              Diaz,A.,Merino,P.,Manrique,LG,Cheng,L.和Yepes ,M.(2019)。尿激酶型纤溶酶原激活剂(uPA)通过ezrin介导的外周星形细胞过程的形成,保护了缺血性脑中的三联突触。Ĵ Cereb血流代谢39(11):2157至2171年。              
Diaz,A.,Merino,P.,Guo,JD,Yepes ,MA,McCann,P.,Katta,T.,Tong,EM,Torre,E.,Rangaraju ,S.和Yepes ,M.(2020)。尿激酶型纤溶酶原激活物从保护可溶性大脑皮层神经甲β诱导的突触损害。Ĵ神经科学40(21):4251-4263。
Halassa ,MM,Fellin ,T。和Haydon,PG(2007)。三方突触:神经胶质传递在健康和疾病中的作用。趋势医学杂志13(2):54-63。
霍林斯沃思(EB),麦克尼尔(McNeal),美国东部(ET),伯顿(Burton),JL,威廉姆斯(RJ),戴利(Daly),JW和克里夫林(Creveling )C.R.(1985)。豚鼠大脑皮层中过滤的突触神经质体制剂的生化特征:环腺苷3':5'-一磷酸生成系统,受体和酶。Ĵ神经科学5(8):2240至2253年。
AS的Horn和OT的Phillipson(1976)。大鼠边缘前脑中的一种去甲肾上腺素敏感的腺苷酸环化酶:激动剂,肾上腺皮质激素和抗精神病药的制备,性质和作用。Eur J Pharmacol 37(1):1-11。
              亨特,加州,申克,LJ和肯尼迪,MB(1996)。PSD-95与前脑突触中的突触后密度相关,而不与突触前膜相关。Ĵ神经科学16(4):1380至1388年。              
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Rao,A。和Steward,O。(1991)。突触后膜特化的蛋白质成分是局部合成的证据:分析突触体内合成的蛋白质。Ĵ神经科学11(9):2881至2895年。              
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Copyright: © 2021 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. Diaz, A. E., Torre, E. and Yepes, M. (2021). Preparation of Synaptoneurosomes to Study the Synapse in the Murine Cerebral Cortex. Bio-protocol 11(2): e3896. DOI: 10.21769/BioProtoc.3896.
  2. Diaz, A., Merino, P., Guo, J. D., Yepes, M. A., McCann, P., Katta, T., Tong, E. M., Torre, E., Rangaraju, S. and Yepes, M. (2020). Urokinase-type plasminogen activator protects cerebral cortical neurons from soluble Aβ-induced synaptic damage. J Neurosci 40(21): 4251-4263.
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Preeti Kute
University of Bergen
Hello,
Thanks for the protocol. You mention that these SN can be stored at -80 for a year. What is the exact application of these SNs? Have you also done an EM to check the integrity of these synaptoneurosomes? Also, people use synaptoneurosomes for synaptic stimulation, have you tried the same after this protocol?
Best,
Preeti
2021/2/12 3:05:28 回复
Ariel Diaz
Yerkes National Primate Research Center Atlanta

Hi Preeti,

Thank you for reading our protocol. These SNs can be used in all of the applications you mentioned; however, if you store them at -80, you will compromise the SNs integrity. If you want to perform functional and structural studies, you should use fresh SNs. Additionally, the EGTA in the buffers can be decreased or removed to reduce the interference with calcium signaling (the protease activity will increase too).

Best,

Ariel

2021/2/19 6:16:35 回复