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Preparation of Synaptosomes from the Motor Cortex of Motor Skill Trained Mice
从经过运动技能培训的小鼠的运动皮质中制备突触体

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Abstract

Learning and memory are thought to occur due to changes in synaptic strength. Strengthening of synapses due to Long Term Potentiation mechanisms are mediated by increases in synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) levels. Here we describe a protocol to isolate and quantify synaptic AMPAR subunit GluA1 levels from the motor cortex of mice which have undergone motor skill training.

Keywords: Synaptosome(突触小体), Motor cortex(运动皮层), Slices(片)

Materials and Reagents

  1. C57BL6 mouse (Mus musculus)
  2. Isoflurane (Isothesia) (Butler animal supplies)
  3. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: S-271 )
  4. Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: P217-500 )
  5. Sodium bicarbonate (NaHCO3) (Thermo Fisher Scientific, catalog number: S233-500 )
  6. Monosodium phosphate (NaH2PO4) (Thermo Fisher Scientific, catalog number: S369-500 )
  7. Magnesium sulphate heptahydrate (MgSO4.7H2O) (Thermo Fisher Scientific, catalog number: MG63-500 )
  8. Calcium chloride (CaCl2) (Thermo Fisher Scientific, catalog number: C79-500 )
  9. Dextrose (Thermo Fisher Scientific, catalog number: D16-500 )
  10. Sucrose (Thermo Fisher Scientific, catalog number: S5-3 )
  11. Radio immunoprecipitation assay (RIPA) lysis buffer (see Recipes)
  12. Roche complete protease inhibitor cocktail pellets (Roche Diagnostics, catalog number: 11697498 001 )
  13. Protease inhibitor (Sigma-Aldrich, catalog number: p8340 )
  14. Phosphatase inhibitor (Sigma-Aldrich, catalog number: 3 p0044 )
  15. Krazy glue
  16. Millipore anti GluA1 antibody(Millipore, catalog number: ab1504 )
  17. Cell signaling anti-rabbit HRP antibody (Cell Signaling Technology, catalog number: 7074S )
  18. Agar powder (Alfa Aesar, catalog number: A10752 )
  19. Artificial cerebrospinal fluid (ASCF) (see Recipes)
  20. High magnesium ACSF (see Recipes)
  21. Agar block (see Recipes)
  22. Sucrose media (see Recipes)

Equipment

  1. Leica vibratome S1000
  2. Scissors (one large and one small)
  3. Forceps
  4. Sharp blade
  5. Vibratome injector blade (Leica)
  6. 1.5 ml Eppendorf tube
  7. Pellet pestle motor hand held homogenizer (Kontes, catalog number: 749540-0000 )
  8. 4 ºC table top centrifuge
  9. Ruler
  10. Aerobic air mixture (Lindweld Alloy, model: MAA140 )
  11. Square petri dish with Grid (Thermo Fisher Scientific, catalog number: FB0875711A )
  12. Sonicator (Thermo Fisher Scientific)

Procedure

Note: Animals are trained on a relevant behavioral paradigm. For our experiments we used a motor forelimb reaching task (Padmashri et al., 2013). The technique described may be adopted to evaluate synaptic changes in any brain region relevant to a specific behavioral paradigm.

  1. Synaptosome preparation
    1. Prepare fresh artificial cerebrospinal fluid (1x ACSF) and bubble with aerobic air mixture at room temperature for at least 15 min.
    2. Prepare a 4 mM MgSO4 ACSF (high Mg2+ ACSF) and keep in an ice bath with constant bubbling.
    3. Anesthetize the animal using isoflurane gas and euthanize by decapitation.
    4. Make a sharp incision through the skin on top of the skull. Separate the skin from the bone.
    5. Make a straight incision from the Foramen Magna to the top of the skull. Separate out the two sections of the skull to expose the brain.
    6. Wash the brain with about 1 ml cold High Mg2+ ACSF.
    7. Isolate the brain and lay out transversely (Figure 1C). Cut off the cerebellum and the olfactory bulb (Figure 1D). Stand the brain on the caudal side and cut out the ventral portion of the brain (Figure 1E).
    8. Glue the caudal portion of the brain on the vibratome plate using a rectangle piece of agar as support (Figure 1F). Make sure the brain is submerged under ice cold High Mg2+ ACSF during slicing (Figure 1G). High Mg2+ ACSF is used as this prevents excitotoxicty to neurons by blocking N-methyl-D-aspartate receptor (NMDAR).
    9. Make thin coronal sections until the corpus callosum from the two hemispheres join (Figure 1H). Then make two 750 micrometer coronal sections which now contain the primary motor cortex M1 (1 mm anterior and 0.5 mm posterior to the bregma and 0.75 to 2.5 mm lateral).
    10. Incubate the slices in a submersion chamber in ACSF at room temperature for 1 h with constant bubbling.
    11. After incubation, place the brain slice on a petri dish under ASCF and constant bubbling at room temperature. Use a blade to isolate the forelimb region of primary motor cortex by making cuts 0.75 and 2.5 mm lateral to the midline (Figure 1I).
    12. Place the brain tissue into a 1.5 ml Eppendorf tube containing 300 µl of 0.32 M sucrose media 1 (see Recipes) containing protease inhibitor (1 tablet in 50 ml of media).
    13. Homogenize with a hand held homogenizer for one minute with up and down movements.
    14. Spin down the homogenized sample at 1,500 rpm for 10 min at 4 ºC. The pellet obtained contains nuclear and cellular debris and is discarded.
    15. Collect the supernatant which contains suspended synaptosomes and spin at 13,500 rpm at 4 °C for 20 min.
    16. The pellet obtained is the required synaptosomal preparation.
      Note: The preparation obtained is an approximately 70% enriched preparation of synaptosomes. The preparation has contamination with mitochondria and debris from endoplasmic reticulum organelles. To prepare a more purified form of synaptosomes this preparation will have to be passed through a sucrose or percoll gradient and has not been described in this protocol.


      Figure 1. Preparation of brain slices on a vibratome


      Figure 2. Electron microscopy image of a synaptosome with pre and postsynaptic component obtained from the protocol described (scale bar 1 micrometer)

  2. To evaluate synaptic GluA1 changes
    1. Aspirate out the supernatant from step A14.
    2. Re-suspend the pellet in 200 µl of RIPA lysis buffer.
    3. Lyse the preparation by sonication on ice (no post lysis spin required as the cellular debris is insignificant).
    4. Estimate the protein levels by relevant protein estimation protocols (BCA assay from Pierce).
    5. Run the protein lysates using a standard western blotting technique. We have run 20 microgram of protein for our experiments.
    6. To immunoblot use Millipore anti GluA1 at 1:1,500 dilution as primary antibody and Cell signaling anti-rabbit HRP as secondary antibody at 1:5,000 dilution.

Representative data



Figure 3. Representative western blots of synaptic GluA1 from forelimb M1 regions of trained (tr) and untrained (utr) hemispheres of motor skill trained mice. With motor skill training there is an increase in synaptic GluA1 in the forelimb region of the motor cortex following training. For quantification, GluA1 levels were normalized to GAPDH.

Note: We have evaluated other proteins such as mTOR, ERK, PSD-95, GLuA2, MAP2 from these preparations.

Recipes

  1. Artificial cerebrospinal fluid (ASCF)
    Chemical
    Concentration (mM)
    g/L
    Sodium chloride (NaCl)
    126
    7.365
    Potassium chloride (KCl)
    3
    0.2235
    Monosodium phosphate (NaH2PO4)
    1.25
    0.1725
    Sodium bicarbonate (NaHCO3)
    26
    2.185
    Magnesium sulphate heptahydrate (MgSO4.7H2O)
    1
    0.120
    Calcium chloride dihydrate
    (CaCl2.2H2O)
    2
    0.294
    Dextrose
    10
    1.8
    Water
    Make up to 1 L

    pH
    7.4

    Tonicity
    300 mmoles/kg


    Note: We prepare a 10x stock of ACSF excluding MgSO4, CaCl2 and dextrose and store at 4 °C. Before the experiment a 1x ACSF is prepared and Dextrose, magnesium and calcium salts are added. Both MgSO4 and CaCl2 are prepared and stored as 1 M stocks which are then added in required volume to the 1x ACSF. A point to note is that 1x ACSF is vigorously bubbled for at least 15 min prior to adding the CaCl2 to prevent calcium precipitation. Another alternative is to add the CaCl2 in small volumes with vigorous stirring on a stir plate.

  2. High magnesium ACSF
    Chemical


    ACSF
    1x solution
    1 L
    Magnesium sulphate heptahydrate (MgSO4.7H2O)
    4 mM
    0.984 g/L

    Note: High Magnesium ACSF is used to block NMDA receptors channels and prevent cell death due to excitotoxicty. The Mg2+ is prepared as a 1 M stock and 0.15 ml of 1 M MgSO4 stock is added to 50 ml of previously prepared 1x ACSF to obtain a final concentration of 4 mM of Mg2+.

  3. Agar block
    Chemical
    Weight (in g)
    Agar powder
    33.2
    Sodium chloride
    9
    Water
    Make up to 1 L

    Disperse the NaCl and Agar powder in water. Heat in a microwave oven until all the agar dissolves and a clear solution is obtained. Pour into a square petri dish and allow to cool to get agar blocks. Make small rectangles from the block and stick the breadth of the rectangle on the vibratome plate.

  4. Sucrose media 1
    Solution
    Volume
    2.5 M sucrose
    6.4 ml
    0.5 M HEPES (pH 7.5)
    500 µl
    0.5 M EDTA (pH 8.0)
    10 µl
    Water
    43.09 ml

    Sucrose media is stored at -20 °C. Roche Protease inhibitor cocktail tablet is added to the media before use (1 tablet in 50 ml).

  5. RIPA lysis buffer
    Ingredient
    Weight or volume
    Tris.HCl
    0.3 g
    Sodium chloride
    0.44 g
    Sodium dodecylsulphate
    0.05 g
    Sodium deoxycholate
    0.125 g
    Triton X 100
    0.1 ml
    0.5 M EDTA
    0.05 ml
    Water
    Make up to 50 ml
    pH
    7.4

    Note: Lysis buffer is stored at room temperature. Before usage protease and phosphatase inhibitor are added (10 µl to 1 ml of lysis buffer). The lysis buffer is stored at 4 °C and used within 2 days of preparation.

Acknowledgments

This work was supported by a National Institute of Child Health and Human Development R01 HD67218 (to A. D.). We thank Dr. Gurudutt Pendyala for his valuable insights in establishing the protocol and Dr. Padmashri Ragunathan for comments on the manuscript. The protocol described is modified from previous publications (Weiler et al., 1981) to measure synaptic protein changes in discrete regions of the mouse cortex with behavior.

References

  1. Padmashri, R., Reiner, B. C., Suresh, A., Spartz, E. and Dunaevsky, A. (2013). Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome. J Neurosci 33(50): 19715-19723.
  2. Weiler, M. H., Gundersen, C. B. and Jenden, D. J. (1981). Choline uptake and acetylcholine synthesis in synaptosomes: investigations using two different labeled variants of choline. J Neurochem 36(5): 1802-1812.

简介

认为学习和记忆由于突触强度的变化而发生。 由于长期增强机制引起的突触的加强由突触型α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPAR)水平的增加介导。 在这里,我们描述了一个方法来分离和量化来自运动技能训练的小鼠的运动皮质的突触AMPAR亚基GluA1水平。

关键字:突触小体, 运动皮层, 片

材料和试剂

  1. C57BL6小鼠(Mus musculus)
  2. 异氟烷(Isothesia)(Butler动物用品)
  3. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S-271)
  4. 氯化钾(KCl)(Thermo Fisher Scientific,目录号:P217-500)
  5. 碳酸氢钠(NaHCO 3)(Thermo Fisher Scientific,目录号:S233-500)
  6. 磷酸一钠(NaH 2 PO 4)(Thermo Fisher Scientific,目录号:S369-500)
  7. 硫酸镁七水合物(MgSO 4·7H 2 O 7H 2 O)(Thermo Fisher Scientific,目录号:MG63-500)
  8. 氯化钙(CaCl 2)(Thermo Fisher Scientific,目录号:C79-500)
  9. 右旋糖(Thermo Fisher Scientific,目录号:D16-500)
  10. 蔗糖(Thermo Fisher Scientific,目录号:S5-3)
  11. 放射免疫沉淀测定(RIPA)裂解缓冲液(参见配方)
  12. Roche完全蛋白酶抑制剂混合物颗粒(Roche Diagnostics,目录号:11697498001)
  13. 蛋白酶抑制剂(Sigma-Aldrich,目录号:p8340)
  14. 磷酸酶抑制剂(Sigma-Aldrich,目录号:3 p0044)
  15. 疯狂胶水
  16. Millipore抗GluA1抗体(Millipore,目录号:ab1504)
  17. 细胞信号抗兔HRP抗体(Cell Signaling Technology,目录号:7074S)
  18. 琼脂粉(Alfa Aesar,目录号:A10752)
  19. 人工脑脊液(ASCF)(见配方)
  20. 高镁ACSF(参见配方)
  21. 琼脂块(见配方)
  22. 蔗糖培养基(参见食谱)

设备

  1. 徕卡vibratome S1000
  2. 剪刀(一大一小)
  3. 镊子
  4. 锋利刀片
  5. Vibratome喷油器叶片(Leica)
  6. 1.5 ml Eppendorf管
  7. 颗粒杵电机手持式匀浆器(Kontes,目录号:749540-0000)
  8. 4ºC台式离心机
  9. 标尺
  10. 有氧空气混合物(Lindweld Alloy,型号:MAA140)
  11. 网格方形培养皿(Thermo Fisher Scientific,目录号:FB0875711A)
  12. 超声波仪(Thermo Fisher Scientific)

程序

注意:对动物进行相关行为模式的训练。 对于我们的实验,我们使用电机前肢达成任务(Padmashri等,2013)。 可以采用所描述的技术来评估与特定行为范例相关的任何脑区域中的突触变化。

  1. 突触体准备
    1. 准备新鲜的人工脑脊液(1x ACSF)和泡泡 好氧空气混合物在室温下至少15分钟
    2. 制备4mM MgSO 4 ACSF(高Mg 2+和ACSF),并保持在恒定鼓泡的冰浴中。
    3. 麻醉动物使用异氟烷气体和安乐死通过断头
    4. 做一个锋利的切口通过皮肤顶部的头骨。 将皮肤与骨骼分开。
    5. 从Foramen Magna到顶部做一个直线切口 头骨。 分开颅骨的两个部分暴露大脑。
    6. 用约1ml冷的高Mg 2+ + ACSF清洗脑。
    7. 隔离大脑和横向布局(图1C)。 切断 小脑和嗅球(图1D)。 站在大脑上 尾侧并切除脑的腹侧部分(图1E)。
    8. 使用a胶粘在vibratome板上的大脑的尾部 长方形琼脂作为支持物(图1F)。 确保大脑是 在切片期间(图1G)浸没在冰冷的高Mg 2+ + ACSF下。 高 Mg 2 + 使用ACSF,因为这通过阻断防止对神经元的兴奋性毒性   N-甲基-D-天冬氨酸受体(NMDAR)。
    9. 使薄冠状 直到来自两个半球的胼heres体连接(图1)   )。 然后做两个750微米冠状切片现在包含 主运动皮质M1(前1毫米和后0.5毫米 前囟和0.75至2.5毫米侧)
    10. 在室温下在ACSF的浸没室中孵育切片1小时,同时恒定鼓泡
    11. 孵育后,将脑切片放在ASCF下的培养皿中 并在室温下恒定鼓泡。 使用刀片隔离 前肢区域的主运动皮层通过切割0.75和2.5毫米 横向于中线(图1I)。
    12. 将脑组织放入   1.5ml Eppendorf管,含有300μl0.32M蔗糖培养基1(见 含有蛋白酶抑制剂(1片,在50ml培养基中))
    13. 用手持式匀浆器匀化一分钟,上下运动。
    14. 在4℃下以1,500rpm旋转均质化的样品10分钟。 获得的沉淀物含有核和细胞碎片 舍弃。
    15. 收集含有悬浮的突触体的上清液,并在4℃下以13,500rpm旋转20分钟。
    16. 获得的丸是所需的突触体制剂。
      注意:所获得的制剂是大约70%富集的 突触体的制备。 该制剂有污染 线粒体和来自内质网细胞器的碎片。 至 准备更准确的形式的突触体这个准备将有 通过蔗糖或percoll梯度,并且还没有


      图1.在vibratome上制备脑切片


      图2.具有pre和的突触体的电子显微镜图像 从所述方案获得的突触后组分(比例尺1   微米)

  2. 评估突触GluA1变化
    1. 吸出步骤A14的上清液。
    2. 将沉淀重悬在200μlRIPA裂解缓冲液中
    3. 通过在冰上超声处理制备物(由于细胞碎片不显着,不需要裂解后旋转)
    4. 通过相关蛋白质估计方案(来自Pierce的BCA测定)估计蛋白质水平
    5. 运行蛋白质裂解物使用标准的蛋白质印迹 技术。 我们为我们的实验运行20微克蛋白质。
    6. 对于免疫印迹,使用Millipore抗GluA1以1:1,500稀释 一级抗体和细胞信号抗兔HRP作为次级 抗体以1:5,000稀释。

代表数据



图3.来自运动技能训练的小鼠的训练(tr)和未训练(utr)半球的前肢M1区域的突触GluA1的代表性蛋白质印迹。在运动技能训练中,突触GluA1在 前肢运动皮层区域训练后。 为了定量,将GluA1水平标准化为GAPDH。

注意:我们已经评估了这些制剂中的其他蛋白质,如mTOR,ERK,PSD-95,GLuA2,MAP2。

食谱

  1. 人工脑脊液(ASCF)
    化学品
    浓度(mM):
    g/L
    氯化钠(NaCl)
    126
    7.365
    氯化钾(KCl)
    3
    0.2235
    磷酸一钠(NaH 2 PO 4)
    1.25
    0.1725
    碳酸氢钠(NaHCO 3)
    26
    2.185
    硫酸镁七水合物(MgSO 4)7H/7H 2 O)
    1
    0.120
    氯化钙二水合物
    (CaCl 2 2H O)
    2
    0.294
    葡萄糖
    10
    1.8

    弥补1 L

    pH
    7.4

    强度
    300 mmoles/kg


    /em> 和右旋糖,并在4°C储存。 在实验之前,制备1×ACSF,并加入葡萄糖,镁和钙盐。 准备了两个MgSO 4 和CaCl 2 并作为1M储备储存,然后以所需体积加入1x ACSF。 需要注意的是,1x ACSF是有力的 鼓泡至少15分钟,然后加入CaCl 2 以防止钙沉淀。 另一种方法是在剧烈搅拌下在搅拌盘上小量加入CaCl 2 。
  2. 高镁ACSF
    化学品


    ACSF
    1x解决方案
    1 L
    硫酸镁七水合物(MgSO 4)7H/7H 2 O)
    4 mM
    0.984 g/L

    注意:高镁ACSF用于阻断NMDA受体通道并防止由于兴奋性毒性引起的细胞死亡。 将Mg 2 + 制备为1M储备液和0.15ml 1M Mg 4 将终浓度为4mM的Mg sup> 。

  3. 琼脂块
    化学品
    重量(g)
    琼脂粉
    33.2
    氯化钠
    9

    弥补1 L

    将NaCl和琼脂粉末分散在水中。 在微波炉中加热,直到所有的琼脂溶解并得到澄清的溶液。 倒入方形培养皿,让其冷却,得到琼脂块。 从块中制作小矩形,并将矩形的宽度粘贴在vibratome板上。

  4. 蔗糖培养基1
    解决方案

    2.5 M蔗糖 6.4 ml
    0.5 M HEPES(pH 7.5)
    500微升
    0.5 M EDTA(pH 8.0)
    10微升

    43.09毫升

    蔗糖培养基储存在-20℃。 Roche蛋白酶抑制剂混合物片剂在使用前加入到培养基中(1片在50ml中)
  5. RIPA裂解缓冲液
    成分
    重量或体积
    Tris HCl
    0.3 g
    氯化钠
    0.44克
    十二烷基硫酸钠
    0.05 g
    脱氧胆酸钠
    0.125克
    Triton X 100
    0.1 ml
    0.5 M EDTA
    0.05 ml

    补足至50ml ml
    pH
    7.4

    注意:裂解缓冲液在室温下储存。 在使用前,加入蛋白酶和磷酸酶抑制剂(10μl至1ml裂解缓冲液)。 裂解缓冲液储存在4℃下,并在制备后2天内使用。

致谢

这项工作得到国家儿童健康和人类发展研究所R01 HD67218(A.D.)的支持。我们感谢Gurudutt Pendyala博士在建立协议方面的宝贵见解,以及Padmashri Ragunathan博士对手稿的意见。描述的方案从以前的出版物(Weiler等人,1981)修改以测量具有行为的小鼠皮质的离散区域中的突触蛋白质变化。

参考文献

  1. Padmashri,R.,Reiner,B.C.,Suresh,A.,Spartz,E.and Dunaevsky,A。(2013)。 改变结构和功能突触可塑性与运动技能学习在脆弱X综合征的小鼠模型。 a> J Neurosci 33(50):19715-19723。
  2. Weiler,M.H.,Gundersen,C.B。和Jenden,D.J。(1981)。 突触体中的胆碱吸收和乙酰胆碱合成:使用两种不同标记的胆碱变体进行的研究。 J Neurochem 36(5):1802-1812。
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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。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Suresh, A. and Dunaevsky, A. (2015). Preparation of Synaptosomes from the Motor Cortex of Motor Skill Trained Mice. Bio-protocol 5(4): e1398. DOI: 10.21769/BioProtoc.1398.
  2. Padmashri, R., Reiner, B. C., Suresh, A., Spartz, E. and Dunaevsky, A. (2013). Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome. J Neurosci 33(50): 19715-19723.
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