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Synaptoneurosome Preparation from C57BL/6 Striata
从C57BL/6 小鼠纹状体制备突触神经小体   

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Abstract

Activity-dependent local mRNA translation endows synapses to remodel their structure and function (Bramham and Wells, 2007). This process is tightly controlled by the state of phosphorylation of several components of the translational machinery including initiation factors and ribosomal proteins (Buffington et al., 2014). The present protocol describes a method to prepare striatal synaptoneurosomes, from adult mice, containing both pre- and postsynaptic elements in which the level of synaptic phospho-proteins can be quantified (Biever et al., 2015).

Keywords: Synaptoneurosome(突触神经小体), Striatum(纹状体), Phospho-protein(磷酸化蛋白)

Materials and Reagents

  1. 1 ml dounce tissue grinder (Capitol scientific, Wheaton®, catalog number: 357538 )
  2. 5 ml and 60 ml syringes (BD, Plastipak, catalog number: 309647 and 300866 , respectively)
  3. Nylon net filters 100 μm (Merck Millipore Corporation, catalog number: NY1H02500 )
  4. Mitex membrane filter 10 μm (Merck Millipore Corporation, catalog number: LCWP02500 )
  5. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 00 30120086 )
  6. C57BL/6 mice (≥8 weeks old, male or female) (Mus musculus)
  7. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  10. Potassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P3786 )
  11. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: 71630 )
  12. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  13. D-(+)-Glucose (Sigma-Aldrich, catalog number: 67528 / G6728 )
  14. HEPES (pH 7.4) (Sigma-Aldrich, catalog number: H3375 )
  15. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  16. Sodium fluorate (Sigma-Aldrich, catalog number: S7920 )
  17. Sodium pyrophosphate decahydrate (Sigma-Aldrich, catalog number: 221368 )
  18. Glycerol phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G6501 )
  19. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  20. Leupeptin hydrochloride (Sigma-Aldrich, catalog number: L0649 )
  21. Pepstatin (Sigma-Aldrich, catalog number: P4265 )
  22. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: 78830 )
  23. Phospho-S845-GluR1 [Anti-phospho-GluR1 (Ser845) Antibody, clone EPR2148, rabbit monoclonal] (Merck Millipore Corporation, catalog number: 04-1073 )
  24. Phospho-T185/Y187-ERK2 [Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)] (Cell Signaling Technology, catalog number: 9101 )
  25. Phospho-S235/236-rpS6 (Cell Signaling Technology, catalog number: 2211 )
  26. Phospho-S209-eIF4E (Cell Signaling Technology, catalog number: 9741 )
  27. β-actin [AC-15] (Abcam, catalog number: AB6276 )
  28. Synaptoneurosome buffer (see Recipes)

Equipment

  1. Swinnex filter holder (Merck Millipore Corporation, catalog number: SX0002500 )
  2. 4 ºC Eppendorf table centrifuge

Procedure

Note: This technique has been used to evaluate biochemical changes after pharmacological treatments in synaptoneurosome preparations of the striatum (Biever et al., 2015). However, the same protocol can be applied to other brain regions or after the performance of behavioral paradigms.

  1. Kill the mouse by cervical dislocation or decapitation and rapidly immerse the head in liquid nitrogen for 4 sec to rapidly cool down the brain without allowing it to freeze (this step helps for the brain dissection and to better preserve protein phosphorylation). We recommend to euthanize the mouse with physical methods such as cervical dislocation rather than using anesthetics or carbon dioxide, but this step should be adapted to local ethical committee guidelines.
  2. Remove the brain, remove the meninges, and dissect the striatum of the 2 hemispheres (≈15-20 mg) on an ice-cooled glass dish as it is shown in Video 1.

    Video 1. Striatum extraction

  3. Homogenize the striata in a glass dounce homogenizer (10 strokes of the loose pestle followed by 10 strokes of the tight pestle) with 1 ml of synaptoneurosome buffer kept on ice. The number of strokes might vary depending on the individual homogenizer/pestles and the size of the brain area. All the steps are performed at 4 °C (either on ice or in a 4 °C room). Representative pictures of the homogenates at each step are shown in Figure 1.
  4. Transfer the homogenate into an Eppendorf tube and aliquot 100 μl as an input.
  5. Prepare all the material required for the synaptoneurosome preparation procedure as indicated in Figure 2.
  6. Pre-wet 3 Nylon net filters (100 μm pore) in synaptoneurosome buffer on ice.
  7. Attach a 5 ml syringe without the plunger (syringe #1) to a swinnex filter holder (#1) containing the 3 pre-wetted Nylon net filters (100 μm), as illustrated in Figure 3. Then, attach this swinnex to another 5 ml syringe (syringe #2) without the plunger, which has been attached at the dispensing tip end to a swinnex filter holder containing 1 Mitex membrane filter (10 μm pore) (swinnex filter holder #2). Place on the other side of the swinnex #2 a 1.5 ml Eppendorf tube on ice to collect the sample.
  8. With a 1-ml pipette, load the sample into the end of the barrel of the syringe #1 attached to a swinnex filter holder #1, insert the plunger and push down on the plunger until all the air is gone from the syringe.
  9. Remove the 5 ml syringe #1 and replace it by a 60 ml syringe with the plunger being pulled back. Once attached to the swinnex filter holder #1 containing the 3 Nylon net filters (100 μm), push down on the plunger until all the air is gone from the syringe. This step helps to push down the sample to the barrel of syringe #2.
  10. Remove the swinnex filter holder #1 containing the 3 Nylon net filters (100 μm), place the plunger of the 5 ml syringe #2, which is connected to the swinnex filter holder #2 containing 1 Mitex membrane filter (10 μm), and push down until all the air is gone from the syringe.
  11. Remove the syringe #2 and replace it with a 60 ml syringe with the plunger being pulled back. Once attached to the swinnex filter holder #2, push down on the plunger until all the air is gone from the syringe. At this step, the homogenate should be in the 1.5 ml Eppendorf tube placed on ice.
  12. Centrifuge 1 min at 4,000 x g at 4 °C. Pipette the supernatant into a new Eppendorf tube and discard the pellet. A representative image of how the pellet looks is shown in Figure 1b.
  13. Centrifuge the supernatant for 4 min at 14,000 x g at 4 °C. Discard the supernatant and resuspend the pellet in 100 μl synaptoneurosome buffer. A representative image of how the pellet looks is shown in Figure 1c. (The volume to re-suspend the pellet might change depending on the protein concentration desired. From the striata of one mouse the synaptoneurosome preparation obtained has a protein concentration around 1-2 μg/μl). The pellet can be stored at -80 °C until used.
    Note: In this type of synaptoneurosome preparation we obtained samples containing enrichment of both presynaptic and postsynaptic markers, a substantial reduction of glial markers and absence of nuclear markers (Biever et al., 2015). Moreover, phospho-proteins can be detected as shown in Figure 4.

Representative data


Figure 1. Representative images of the homogenates at different steps. a. Sample obtained after the homogenization of the striata of one mouse as indicated in step 3. b. Sample obtained after the first centrifugation as indicated in step 12. c. Sample obtained after the second centrifugation as indicated in step 13.


Figure 2. Materials required for synaptoneurosome preparation procedure. Place three pre-wetted Nylon net filters (100 μm) in swinnex filter holder #1 and one Mitex membrane filter (10 μm) in swinnex filter holder #2. Mount the apparatus in the following order from the bottom to the top: Eppendorf on ice < swinnex filter holder #2 < 5 ml syringe #2 without plunger < swinnex filter holder #1 < 5 ml syringe #1 without plunger. After loading the sample with a pipette into the barrel of syringe #1, add the plunger and push down until all the air is gone from the syringe. Remove the 5 ml syringe #1 and replace it by a 60 ml syringe with the plunger being pulled back. Once attached to the swinnex filter holder #1, push down on the plunger until all the air is gone from the syringe. This step helps to push down the sample to the barrel of syringe #2. Continue the protocol as indicated from step 10.


Figure 3. Illustration of the mounted apparatus for synaptoneurosome preparation procedure. A 5 ml syringe (syringe #1) is attached to a swinnex filter holder containing 3 Nylon net filters (100 μm) (swinnex filter holder #1) and connected to another 5 ml syringe (syringe #2), which is attached to a second swinnex filter holder containing a Mitex membrane filter (10 μm) (swinnex filter holder #2). A 1.5 ml Eppendorf tube is placed on ice on the other side of the swinnex to collect the sample that was initially loaded into the syringe #1.
Note: Syringes #1 and #2 could be replaced with syringes of different sizes depending on sample volume.


Figure 4. Representative western blots of phospho-proteins in striatal synaptoneurosome preparations. Ten μg per lane of synaptoneurosomal samples were separated in 13% SDS-polyacrylamide gel before electrophoretic transfer onto Immobilon-P membranes. A regular western blot protocol was used including the following antibodies: phospho-S845-GluR1, phospho-T185/Y187-ERK2, phospho-S235/236-rpS6 phospho-S209-eIF4E and β-actin.

Acknowledgments

This work was supported by Inserm, Agence Nationale de la Recherche (Grant ANR-2010-JCJC-1412 to E. V.), and a NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation (to E. P.). A. B. is supported by the Fonds National de la Recherche, Luxembourg (Grant 3977033). E. P. was a recipient of an EMBO Fellowship with the support of European Commission (Grants EMBOCOFUND2010 and GA-2010-267146) and a Marie Curie Intra-European Fellowship IEF327648.

Recipes

  1. Synaptoneurosome buffer (use sterile water of high purity, such as Milli-Q water)
    2.5 mM CaCl2
    124 mM NaCl
    3.2 mM KCl
    1.06 mM KH2PO4
    26 mM NaHCO3
    1.3 mM MgCl2
    10 mM D-(+)-Glucose
    20 mM HEPES (pH 7.4)
    0.15 μM aprotinin
    11 μM leupeptin
    1.5 μM pepstatin
    0.6 mM phenylmethylsulfonyl fluoride
    1 mM sodium orthovanadate*
    100 mM sodium fluoride*
    5 mM sodium pyrophosphate decahydrate*
    40 mM Glycerol phosphate disodium salt hydrate*
    Note: The buffer should be freshly prepared the day of the experiment. Buffer components marked with an asterisk are only required if phospho-proteins are analyzed in the synaptoneurosome preparations.

References

  1. Biever, A., Puighermanal, E., Nishi, A., David, A., Panciatici, C., Longueville, S., Xirodimas, D., Gangarossa, G., Meyuhas, O., Herve, D., Girault, J. A. and Valjent, E. (2015). PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons. J Neurosci 35(10): 4113-4130.
  2. Bramham, C. R. and Wells, D. G. (2007). Dendritic mRNA: transport, translation and function. Nat Rev Neurosci 8(10): 776-789.
  3. Buffington, S. A., Huang, W. and Costa-Mattioli, M. (2014). Translational control in synaptic plasticity and cognitive dysfunction. Annu Rev Neurosci 37: 17-38.

简介

活动依赖的局部mRNA翻译赋予突触重塑其结构和功能(Bramham和Wells,2007)。 该过程由包括起始因子和核糖体蛋白在内的翻译机理的几个组分的磷酸化状态来严格控制(Buffington等,2014)。 本方案描述了一种从成年小鼠制备纹状体synaptoneurosomes的方法,其含有可以量化突触磷酸蛋白水平的突触前和突触后元件(Biever等,2015)。

关键字:突触神经小体, 纹状体, 磷酸化蛋白

材料和试剂

  1. 1ml dounce组织研磨机(Capitol scientific,Wheaton ,目录号:357538)
  2. 5ml和60ml注射器(BD,Plastipak,目录号:309647和300866)
  3. 尼龙网过滤器100μm(Merck Millipore Corporation,目录号:NY1H02500)
  4. Mitex膜过滤器10μm(Merck Millipore Corporation,目录号:LCWP02500)
  5. 1.5ml Eppendorf管(Eppendorf,目录号:0030120086)
  6. C57BL/6小鼠(≥8周龄,雄性或雌性)(Mus musculus)
  7. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  9. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  10. 磷酸钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P3786)
  11. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:71630)
  12. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  13. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:67528/G6728)
  14. HEPES(pH7.4)(Sigma-Aldrich,目录号:H3375)
  15. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  16. 氟酸钠(Sigma-Aldrich,目录号:S7920)
  17. 焦磷酸钠十水合物(Sigma-Aldrich,目录号:221368)
  18. 甘油磷酸二钠盐水合物(Sigma-Aldrich,目录号:G6501)
  19. 抑肽酶(Sigma-Aldrich,目录号:A1153)
  20. 盐酸亮肽素(Sigma-Aldrich,目录号:L0649)
  21. 胃酶抑素(Sigma-Aldrich,目录号:P4265)
  22. 苯甲基磺酰氟(Sigma-Aldrich,目录号:78830)
  23. Phospho-S845-GluR1 [抗磷酸-GluR1(Ser845)抗体,克隆EPR2148,兔单克隆](Merck Millipore Corporation,目录号:04-1073)
  24. 磷酸-T185/Y187-ERK2 [Phospho-p44/42MAPK(Erk1/2)(Thr202/Tyr204)](Cell Signaling Technology,目录号:9101)
  25. Phospho-S235/236-rpS6(Cell Signaling Technology,目录号:2211)
  26. Phospho-S209-eIF4E(Cell Signaling Technology,目录号:9741)
  27. β-肌动蛋白[AC-15](Abcam,目录号:AB6276)
  28. 突触单体缓冲液(参见配方)

设备

  1. Swinnex过滤器保持器(Merck Millipore Corporation,目录号:SX0002500)
  2. 4oCEppendorf台式离心机

程序

注意:这种技术已经用于评估在纹状体的synaptoneurosome制剂中的药理学治疗后的生物化学变化(Biever等人,2015)。然而,相同的协议可以应用于其他大脑区域或行为范式之后。

  1. 通过颈椎脱位或断头杀死小鼠,并迅速将头部浸泡在液氮中4秒,以快速冷却大脑,而不允许它冻结(这一步有助于大脑解剖和更好地保持蛋白磷酸化)。我们建议使用物理方法例如颈椎脱臼,而不是使用麻醉剂或二氧化碳来安乐死小鼠,但这一步应该适应当地的伦理委员会指南。
  2. 去除大脑,去除脑膜,并在冰冷的玻璃盘上剖开2个半球的纹状体(≈15-20mg),如视频1所示。

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    视频1.纹理提取
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  3. 在玻璃dounce匀浆器(10个松散杵,随后紧10杵的紧杵)与1毫升synaptoneurosome缓冲液保持在冰上均化纹理。中风的数量可以根据个体均化器/杵和脑区的大小而变化。所有步骤在4℃下进行(在冰上或在4℃室中)。每个步骤的匀浆的代表性图片如图1所示
  4. 将匀浆转移到Eppendorf管中,并等分100μl作为输入
  5. 准备如图2所示的synaptoneurosome准备程序所需的所有材料。
  6. 在冰上的synaptoneurosome缓冲液中预湿3尼龙网过滤器(100μm孔)
  7. 将没有柱塞(注射器#1)的5ml注射器连接到含有3个预润湿的尼龙网过滤器(100μm)的swinnex过滤器保持器(#1),如图3所示。然后,将该swinnex连接到另一个5 ml注射器(注射器#2),其已经在分配末端附接到含有1 Mitex膜过滤器(10μm孔)(swinnex过滤器保持器#2)的swinnex过滤器支架上。将位于swinnex#2另一侧的1.5 ml Eppendorf管置于冰上收集样品。
  8. 用1-ml移液管将样品加载到连接到#1的swinnex过滤器支架#1的注射器#1的筒的末端,插入柱塞并向下推到柱塞上,直到所有空气从注射器排出。 br />
  9. 取出5毫升注射器#1,并用一个60毫升注射器替换它,柱塞被拉回。一旦连接到包含3个尼龙网过滤器(100μm)的swinnex过滤器支架#1,向下推动柱塞,直到所有空气从注射器流出。此步骤有助于将样品下推到#2注射器的筒体
  10. 取出含有3个尼龙网过滤器(100μm)的swinnex过滤器支架#1,将连接到含有1 Mitex膜过滤器(10μm)的swinnex过滤器支架#2的5ml注射器#2的柱塞,向下推直到所有空气从注射器流出。
  11. 取下2号注射器,并用60毫升注射器替换它,柱塞被拉回。一旦连接到#2的swinnex过滤器支架,向下推动柱塞,直到所有的空气从注射器流出。在该步骤中,匀浆应该置于置于冰上的1.5ml Eppendorf管中
  12. 在4℃下以4,000×g离心1分钟。吸取上清液到一个新的Eppendorf管中并丢弃沉淀。图1b示出了粒料如何看起来的代表性图像
  13. 在4℃下以14,000×g离心上清液4分钟。丢弃上清液和重悬在100μlsynaptoneurosome缓冲液沉淀。图1c中示出了粒料如何看起来的代表性图像。 (根据所需的蛋白质浓度,重悬细胞沉淀的体积可能改变。从一只小鼠的纹状体获得的突触单体制剂具有约1-2μg/μl的蛋白质浓度。丸粒可以储存在-80℃直到使用。
    注意:在这种类型的突触单体制备中,我们获得了包含突触前和突触后标记物的富集的样品,胶质标记物的显着减少和不存在核标记物(Biever等人,2015)。此外,可以检测磷蛋白,如图4所示。

代表数据


图1.匀浆在不同步骤的代表性图像。 a。如步骤3所示,一只小鼠纹状体均化后获得的样品。如步骤12所示在第一次离心后获得的样品。第二次离心后获得的样品,如步骤13所示。


在swinnex过滤器支架#1中放置三个预润湿的尼龙网过滤器(100μm),在swinnex过滤器支架#2中放置一个Mitex膜过滤器。以如下顺序从底部到顶部安装设备:在冰上的Eppendorf swinnex过滤器保持器# 5ml注射器#2, swinnex过滤器保持器# 5毫升注射器#1无柱塞。在用移液管将样品装载到#1注射器的筒中之后,添加柱塞并向下推,直到所有空气从注射器排出。取出5毫升注射器#1,并用一个60毫升注射器替换它,柱塞被拉回。一旦连接到swinnex过滤器支架#1,向下推动柱塞,直到所有的空气从注射器流出。该步骤有助于将样品下推到注射器#2的筒体。继续步骤10所示的协议。


图3.用于synaptoneurosome制备程序的安装设备的图示。将5ml注射器(注射器#1)连接到含有3个尼龙网过滤器(100μm)(swinnex过滤器保持器# 1),并连接到另一个5ml注射器(注射器#2),其连接到包含Mitex膜过滤器(10μm)(swinnex过滤器保持器#2)的第二swinnex过滤器保持器。将1.5ml Eppendorf管放置在swinnex的另一侧的冰上,以收集最初装载到#1注射器中的样品。
注意:根据样品量,注射器#1和#2可以用不同尺寸的注射器替换。


图4.纹状体synaptoneurosome制剂中磷蛋白的代表性蛋白质印迹。 在电泳转移到Immobilon-P膜上之前,在13%SDS-聚丙烯酰胺凝胶中分离10μg的每个通道的synaptoneurosomal样品。使用常规的蛋白质印迹方案,包括以下抗体:磷酸-S845-GluR1,磷酸-T185/Y187-ERK2,磷酸-S235/236-rpS6磷酸-S209-eIF4E和β-肌动蛋白。

致谢

这项工作由Inserm,Agence Nationale de la Recherche(授予ANR-2010-JCJC-1412至E.V.)和来自大脑和行为研究基金会(授予E.P.)的NARSAD年轻研究者授予的支持。 A. B.由Fonds National de la Recherche,Luxembourg(Grant 3977033)支持。 E.P.是在欧盟委员会(Grants EMBOCOFUND2010和GA-2010-267146)和Marie Curie欧洲团契IEF327648的支持下的EMBO奖学金的接受者。

食谱

  1. 突触体神经元缓冲液(使用高纯度的无菌水,如Milli-Q水)
    2.5mM CaCl 2·h/v 124mM NaCl 3.2 mM KCl
    1.06mM KH 2 PO 4 sub/
    26mM NaHCO 3/v/v 1.3mM MgCl 2·h/v 10mM D - (+) - 葡萄糖 20mM HEPES(pH7.4) 0.15μM抑肽酶
    11μM亮肽素
    1.5μM胃酶素
    0.6mM苯甲基磺酰氟 1 mM原钒酸钠*
    100mM氟化钠*
    5mM焦磷酸钠十水合物*
    40mM磷酸甘油二钠盐水合物*
    注意:缓冲区应该在实验当天准备好。如果在synaptoneurosome制剂中分析磷蛋白,则只需要用星号标记的缓冲液组分。

参考文献

  1. Biever,A.,Puighermanal,E.,Nishi,A.,David,A.,Panciatici,C.,Longueville,S.,Xirodimas,D.,Gangarossa,G.,Meyuhas,O.,Herve, Girault,JA和Valjent,E。(2015)。 核糖体蛋白S6的PKA依赖性磷酸化与纹状体和纹状体中等大小的翻译效率不相关spiny neurons。 J Neurosci 35(10):4113-4130。
  2. Bramham,C.R。和Wells,D.G。(2007)。 Dendritic mRNA:转运,翻译和功能。 Nat Rev Neurosci < 8(10):776-789。
  3. Buffington,S.A.,Huang,W。和Costa-Mattioli,M。(2014)。 突触可塑性和认知功能障碍的平移控制。 Annu Rev Neurosci < 37:17-38。
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Copyright: © 2016 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. Puighermanal, E., Biever, A. and Valjent, E. (2016). Synaptoneurosome Preparation from C57BL/6 Striata. Bio-protocol 6(4): e1735. DOI: 10.21769/BioProtoc.1735.
  2. Biever, A., Puighermanal, E., Nishi, A., David, A., Panciatici, C., Longueville, S., Xirodimas, D., Gangarossa, G., Meyuhas, O., Herve, D., Girault, J. A. and Valjent, E. (2015). PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons. J Neurosci 35(10): 4113-4130.
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