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In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling
使用稀疏标记进行体外海马神经元树突棘缩回实验   

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Abstract

Dendritic spines are the post-synaptic structures that play a central role in excitatory synaptic transmission. Developmental spinogenesis relies on a variety of stimuli such as those derived from cell-cell communication and their downstream signaling. Here, we describe an in vitro assay of dendritic spine retraction using hippocampal slice culture, in which individual neurons are sparsely and brightly labeled by the Supernova method, for the study of molecular mechanisms of spine development.

Keywords: Spine(脊柱), Retraction assay(收缩试验), EphA(EphA), Spinogenesis(spinogenesis)

Materials and Reagents

  1. Materials
    1. Electrode (5 mm Φ platinum disk) (Nepa Gene, model: CUY650P5 )
    2. Surgical needle (ELP, model: CR13-50 )
    3. Glass capillary (Warner Instruments, model: GC150TF-10 )
    4. NuncTM culture plate (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
    5. Millicell culture plate inserts (EDM Millipore, catalog number: PICM03050 )
    6. Confetti (LCR membrane filter) (EDM Millipore, catalog number: FHLC01300 )
    7. Petri dish (60 mm) (Sigma-Aldrich, catalog number: Z721034 )
      Note: This product has been discontinued.
    8. Microcentrifuge tube (Sigma-Aldrich, catalog number: Z666505 )
    9. Cover glass (MATSUNAMI GLASS)
    10. Microscope glass slide (MATSUNAMI GLASS)
    11. Aspirator tube assembly (Drummond Scientific, catalog number: 2-000-000 )

  2. Sparse and bright labeling of hippocampal neurons using the in utero electroporation-based Supernova system (Mizuno et al., 2014)
    1. Pregnant mouse (E13.5-E15.5)
    2. Maxi-prep kit
    3. Trypan blue solution (0.4%) (Sigma-Aldrich, catalog number: T8154 )
    4. Supernova vector DNA solution (pK036.TRE-Flpe-WPRE-pA and pK037.CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA)
    5. Somnopentyl (Pentobarbital sodium)
    6. 70% ethanol
    7. Saline

  3. Organotypic hippocampal slice culture
    1. Mouse pup (P4-P5)
    2. MEM with gluramax-1 (Thermo Fisher Scientific, catalog number: 41090-028 )
    3. EBSS (Thermo Fisher Scientific, catalog number: 14155-048 )
    4. D-glucose (NACALAI TESQUE, catalog number: 16805-35 )
    5. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
    6. Nystatin (Thermo Fisher Scientific, GibcoTM, catalog number: 15340029 )
    7. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
    8. Horse serum (Sigma-Aldrich, catalog number: H1270-500 ml )
    9. Culture medium (See Recipes)
    10. Slicing buffer(See Recipes)

  4. In vitro retraction assay
    1. EphrinA3-Fc (R&D Systems, catalog number: BT359 )
    2. Human Fc fragment (Jackson ImmunoResearch, catalog number: 009-000-008 )
    3. Goat anti-human IgG, Fc fragment specific (Jackson ImmunoReserach, catalog number: 109-001-008 )
    4. Paraformaldehyde (NACALAI TESQUE, catalog number: 02890-45 )
    5. PBS (-)
    6. Vectashield mounting media with DAPI (Vector laboratories, catalog number: H-1200 )

Equipment

  1. Sparse and bright labeling of hippocampal neurons using the in utero electroporation-based Supernova system.
    1. Electroporator (Nepa Gene, model: Nepa21 )
    2. Water bath
    3. Puller (NARISHIGE Group, model: PC-10 )
    4. Heating table (Leica Biosystems, model: HI1220 )
    5. Surgical tools (scissor, 12 cm) (Fine Science Tools, catalog number: 14001-12 )
    6. Surgical tools (scissor, 10.5 cm) (Fine Science Tools, catalog number: 14088-10 )
    7. Surgical tools (forceps, 12 cm) (Fine Science Tools, catalog number: 11000-12 )
    8. Surgical tools (forceps, 10 cm) (Fine Science Tools, catalog number: 11050-10 )
    9. Surgical tools (ring forceps, 8.5 cm) (Fine Science Tools, catalog number: 11101-09 )

  2. Organotypic hippocampal slice culture
    1. LinearSlicer (Dosaka, model: PRO7 )
    2. 37 °C, 5% CO2 incubator
    3. Pipette

  3. In vitro retraction assay
    1. 25 °C incubator

  4. Imaging and analysis
    1. Confocal microscope (Leica Biosystems, model: TSC-SP5 )
    2. Imaris filament tracer (Bitplane)

Software

  1. Imaris

Procedure

  1. Sparse and bright labeling of hippocampal neurons using the in utero electroporation-based Supernova system
    Note: Using the Supernova system that enables sparse and bright cell labeling with little background is highly recommended for analyses of fine structures such as spines of individual neurons. The Supernova system is described elsewhere (Mizuno et al., 2014). Briefly, this system uses two vectors, TRE-Flpe-WPRE-pA (pK036) and CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA (pK037). In a small population of neurons that carry both vectors, TRE leakage drives weak but over-threshold Flpe expression from the fist vector, which is followed by removal of the FRT-STOP-FRT cassette in a few copies of the second vector and weak expression of tTA from these copies. Then, only in these sparse neurons, tTA binds with TRE and induces strong Flpe expression, which results in removal of the FRT-STOP-FRT cassette from many copies of the second vector, and finally results in extremely strong RFP expression via the positive feedback. Flpe-based Supernova vectors [TRE-Flpe-WPRE-pA (pK036) and CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA (pK037)] are available from T.I. (tiwasato@nig.ac.jp).
    1. To prepare micropipettes, pull glass capillary using a puller under the following conditions: two-step; heater, 65-75; weight, 1-3. Cut off the tip with forceps for appropriate diameter (about 20 μm).
    2. To prepare DNA solution for the Supernova labeling, purify the plasmid set, TRE-Flpe-WPRE-pA (pK036) and CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA (pK037), using endotoxin free Maxi-prep kit, and mix them to a final concentration of 1 μg/μl of pK036 and 50 ng/μl of pK037.
    3. Aliquot 40 μl of the DNA solution, and add 4 μl of Trypan blue solution (0.4%).
    4. Anesthetize a pregnant mouse (E13.5-E15.5) with pentobarbital sodium through intraperitoneal injection (50 mg/kg in saline).
    5. Place the anesthetized mouse on a working plate, and wash the abdomen with 70% ethanol.
    6. Make an incision at the abdominal midline using scissors, and take out the uterus gently onto the saline-moistened gauze. During the surgery, keep the uterus moist with pre-warmed saline (Figure 1).


      Figure 1. An anesthetized pregnant mouse with embryos (E14.5)

    7. Inject 1-2 μl of the DNA solution into the target ventricle of the embryos using the micropipette with an aspirator tube.
    8. Hold the embryos with electrodes (Figure 2: negative electrode on the injected side of the embryo head), and apply electric pulses [40-50 V, P on 50 msec, P off 950 msec (1 Hz), 5 times].


      Figure 2. Schematic illustration of in utero electroporation. DNA injection side (blue) and the relative position of the electrodes for the hippocampal electroporation.

    9. Place the uterus gently into the abdominal cavity.
    10. Fill the cavity with pre-warmed saline.
    11. Suture the surgical incision using surgical needle and thread.
    12. Place the mouse on a heating table at 37 °C until recovery from anesthesia.

  2. Organotypic hippocampal slice culture
    1. Pour 1 ml of culture medium into each well (6-well plate).
    2. Place a Millicell culture plate insert into each well.
    3. Cut confetti disc using scissors (quarter), and put a piece on each insert.
    4. Place the plate into a 5% CO2 incubator at 37 °C.
    5. Pour 10 ml of slicing buffer into a Falcon tube (50 ml), and keep it on ice.
    6. Pour 5 ml of slicing buffer into a Petri dish (60 mm) on ice.
    7. Euthanize pup (P4-P5) by decapitation using scissors.
    8. Place the brain into a Petri dish containing slicing buffer on ice.
    9. Cut the brain half along the midline after removing the cerebellum.
    10. Glue the brain tissue on the stage of LinearSlicer buffer tray.
    11. Fill the buffer tray with the slicing buffer (4 °C).
    12. Cut a 300 μm hippocampal slices (sagittal sections) using LinearSlicer. Leave the entorhinal cortex attached to the hippocampus (Figure 3).


      Figure 3. Dissection of the hippocampus from the brain. Left: A P5 mouse brain was sagittally sliced by a vibratome. Right: A 300 μm-thick slice and cut position were shown.

    13. Collect the slices in a 50 ml Falcone tube containing slicing buffer (4 °C).
    14. Take a slice from the tube using a pipette, and put it on the center of the confetti on insert.
    15. Remove excess buffer from the insert, and place the plate in a 5% CO2 incubator (37 °C).
    16. Change culture medium every 2 days until retraction assay.

  3. In vitro retraction assay
    1. Mix 10 μg of ephrinA3-Fc/Fc and 45 μg of anti-human Fc antibody in a microcentrifuge tube.
    2. Place the tube into a 25 °C incubator.
    3. After 1 h, place the pre-clustered solution into organotypic hippocampal slice culture medium. The final concentration for ephrinA3-Fc/Fc is 10 μg/ml.
    4. Place the plate into a 5% CO2 incubator at 37 °C.
    5. After 16 h, fix the hippocampal slices on the confetti discs with 4% PFA in PBS for 30 min at room temperature.
    6. Mount hippocampal slices onto a glass slide with Vectashield mounting media, and cover with a cover glass.

  4. Imaging and analysis
    1. Image dendritic spines in primary/secondary dendrites of hippocampal pyramidal neurons in CA1 region using a confocal microscope. Sequential z-images consisted of optical section (1,024 x 1,024 pixels) with 0.1 μm intervals using 63x oil immersion objective (numerical aperture, 1.3) with 9x digital zoom. Figure 4 shows z-stacked images of typical dendritic spines.
    2. Reconstruct z-images, and measure spine density, spine length, volume by the Imaris Filament Tracer.


      Figure 4. Example of results of dendritic spine retraction assay. Segments of dendrites from hippocampal slices, after treatment with ephrinA3-Fc (right) or Fc (left). Scale bar: 10 μm.

Recipes

  1. Culture medium
    250 ml MEM with glutamax-1
    125 ml horse serum
    120 ml EBSS with 3% D-glucose
    5 ml penicillin-streptomycin
    0.3 ml nystatin
  2. Slicing buffer
    487.5 ml EBSS
    12.5 ml 1 M HEPES

Acknowledgments

This protocol was adopted from Iwata et al. (2015). This work was supported by the JSPS KAKENHI (15H04263, 16K14559) and MEXT KAKENHI (15H01454).

References

  1. Iwata, R., Matsukawa, H., Yasuda, K., Mizuno, H., Itohara, S. and Iwasato, T. (2015). Developmental RacGAP α2-chimaerin signaling is a determinant of the morphological features of dendritic spines in adulthood. J Neurosci 35(40): 13728-13744.
  2. Mizuno, H., Luo, W., Tarusawa, E., Saito, Y. M., Sato, T., Yoshimura, Y., Itohara, S. and Iwasato, T. (2014). NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates. Neuron 82(2): 365-379.

简介

树突状棘是突触后结构,在兴奋性突触传播中起重要作用。 发育性的发生依赖于各种刺激物,如来自细胞 - 细胞通讯及其下游信号传导的刺激。 在这里,我们描述了使用海马片段培养的树突状脊柱退缩的体外测定,其中单个神经元被Supernova方法稀疏和明亮地标记,用于研究脊柱发育的分子机制。

关键字:脊柱, 收缩试验, EphA, spinogenesis

材料和试剂

  1. 材料
    1. 电极(5mmΦ铂盘)(Nepa Gene,型号:CUY650P5)
    2. 手术针(ELP,型号:CR13-50)
    3. 玻璃毛细管(Warner Instruments,型号:GC150TF-10)
    4. Nunc TM培养板(6孔)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:140675)。
    5. Millicell培养板插入物(EDM Millipore,目录号:PICM03050)
    6. Confetti(LCR膜过滤器)(EDM Millipore,目录号:FHLC01300)
    7. 培养皿(60mm)(Sigma-Aldrich,目录号:Z721034)
      注意:此产品已停产。
    8. 微量离心管(Sigma-Aldrich,目录号:Z666505)
    9. 保护玻璃(MATSUNAMI GLASS)
    10. 显微镜载玻片(MATSUNAMI GLASS)
    11. 抽吸管组件(Drummond Scientific,目录号:2-000-000)

  2. 使用基于子宫内电穿孔的Supernova系统中的海马神经元的稀疏和明亮的标记(Mizuno等人,2014)
    1. 怀孕老鼠(E13.5-E15.5)
    2. Maxi-prep套件
    3. 台盼蓝溶液(0.4%)(Sigma-Aldrich,目录号:T8154)
    4. Supernova载体DNA溶液(pK036.TRE-Flpe-WPRE-pA和pK037.CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA)
    5. 戊巴比妥(戊巴比妥钠)
    6. 70%乙醇
    7. 盐水

  3. 器官型海马切片培养
    1. 鼠标啄(P4-P5)
    2. MEM与gluramax-1(Thermo Fisher Scientific,目录号:41090-028)
    3. EBSS(Thermo Fisher Scientific,目录号:14155-048)
    4. D-葡萄糖(NACALAI TESQUE,目录号:16805-35)
    5. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)
    6. 制霉菌素(Thermo Fisher Scientific,Gibco TM ,目录号:15340029)
    7. HEPES(1μM)(Thermo Fisher Scientific,Gibco TM ,目录号:15630-080)
    8. 马血清(Sigma-Aldrich,目录号:H1270-500ml)
    9. 培养基(见配方)
    10. 切片缓冲区(请参阅配方)

  4. 体外回缩测定
    1. EphrinA3-Fc(R& D Systems,目录号:BT359)
    2. 人Fc片段(Jackson ImmunoResearch,目录号:009-000-008)
    3. 山羊抗人IgG,Fc片段特异性(Jackson ImmunoReserach,目录号:109-001-008)
    4. 多聚甲醛(NACALAI TESQUE,目录号:02890-45)
    5. PBS( - )
    6. 使用DAPI(Vector laboratories,目录号:H-1200)的Vectashield封固介质

设备

  1. 稀疏和明亮的标记海马神经元使用在基于电泳的超声系统。
    1. 电穿孔仪(Nepa Gene,型号:Nepa21)
    2. 水浴
    3. Puller(NARISHIGE Group,型号:PC-10)
    4. 加热台(Leica Biosystems,型号:HI1220)
    5. 外科工具(剪刀,12厘米)(Fine Science Tools,目录号:14001-12)
    6. 外科工具(剪刀,10.5cm)(Fine Science Tools,目录号:14088-10)
    7. 手术工具(镊子,12cm)(Fine Science Tools,目录号:11000-12)
    8. 手术工具(镊子,10厘米)(Fine Science Tools,目录号:11050-10)
    9. 手术工具(环形钳,8.5cm)(Fine Science Tools,目录号:11101-09)

  2. 器官型海马切片培养
    1. LinearSlicer(Dosaka,型号:PRO7)
    2. 37℃,5%CO 2培养箱
    3. 移液器

  3. 体外回缩测定
    1. 25℃培养箱

  4. 成像和分析
    1. 共聚焦显微镜(Leica Biosystems,型号:TSC-SP5)
    2. Imaris细丝示踪剂(位平面)

软件

  1. Imaris

程序

  1. 使用基于子宫内电穿孔的Supernova系统中的海马神经元的稀疏和明亮标记
    注意:强烈建议使用Supernova系统,使用稀疏和明亮的细胞标记与少量背景分析精细结构,如个别神经元的刺。超新系统在别处描述(Mizuno等人,2014)。简言之,该系统使用两个载体TRE-Flpe-WPRE-pA(pK036)和CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA(pK037)。在携带两种载体的小群神经元中,TRE渗漏驱动来自第一载体的弱但超过阈值的Flp1表达,随后在几个拷贝的第二载体中去除FRT-STOP-FRT盒,并且弱表达的tTA。然后,仅在这些稀疏的神经元中,tTA与TRE结合并诱导强Flp表达,这导致从第二载体的许多拷贝中去除FRT-STOP-FRT盒,并且最终通过正反馈导致极强的RFP表达。基于Flip的Supernova载体[TRE-Flpe-WPRE-pA(pK036)和CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA(pK037) ( tiwasato@nig.ac.jp )。
    1. 为了制备微量移液器,在以下条件下使用拉片拉玻璃毛细管:两步;加热器,65-75;重量1-3。用适当直径(约20μm)的钳子切掉尖端。
    2. 为了制备用于Supernova标记的DNA溶液,使用不含内毒素的Maxi纯化质粒组,TRE-Flpe-WPRE-pA(pK036)和CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA(pK037) -prep试剂盒,并将其混合至1μg/μlpK036和50ng /μlpK037的终浓度。
    3. 等分40μl的DNA溶液,并加入4μl台盼蓝溶液(0.4%)
    4. 通过腹膜内注射(50mg/kg盐水)麻醉怀孕小鼠(E13.5-E15.5)与戊巴比妥钠。
    5. 将麻醉的鼠标放在工作板上,用70%乙醇洗腹部
    6. 使用剪刀在腹部中线切口,并轻轻地取出子宫到盐水润湿的纱布上。在手术过程中,用预温热的盐水保持子宫湿润(图1)

      图1.麻醉的怀孕小鼠胚胎(E14.5)

    7. 使用带吸气管的微量吸管将1-2μl的DNA溶液注入胚胎的目标心室。
    8. 用电极保持胚胎(图2:在胚胎头的注射侧上的负电极),并施加电脉冲[40-50V,P在50毫秒,P在950毫秒(1Hz),5次] br />

      图2.子宫内电穿孔的示意图。DNA注射侧(蓝色)和海马电穿孔的电极的相对位置。

    9. 将子宫轻轻地放入腹腔。
    10. 用预热的盐水填充空腔。
    11. 使用手术针和线缝合外科切口
    12. 将鼠标放在37℃的加热台上,直到麻醉恢复
  2. 器官型海马切片培养
    1. 将1ml培养基倒入每个孔(6孔板)
    2. 将Millicell培养板插入每个孔。
    3. 使用剪刀(四分之一)切割五彩纸屑,并在每个插入件上放一块
    4. 将板置于37℃的5%CO 2培养箱中
    5. 将10ml切片缓冲液倒入Falcon管(50ml)中,并保持在冰上
    6. 在冰上将5ml切片缓冲液倒入培养皿(60mm)中
    7. 使用剪刀斩首安乐死小狗(P4-P5)。
    8. 将大脑放入含有在冰上的切片缓冲液的培养皿中
    9. 摘除小脑后沿中线切开大脑一半。
    10. 在LinearSlicer缓冲盘的台上粘贴脑组织。
    11. 使用切片缓冲液(4°C)填充缓冲液托盘。
    12. 切割300微米海马切片(矢状切片)使用LinearSlicer。 留下内嗅皮层附着海马(图3)。


      图3.来自大脑的海马的解剖。左图:通过vibratome对P5小鼠脑进行矢状切片。 右:示出了300μm厚的切片和切割位置
    13. 收集切片在含有切片缓冲液(4℃)的50ml Falcone管中
    14. 使用移液管从管中取一片,并将其放在插入的五彩纸屑的中心
    15. 从插入物中除去过量的缓冲液,并将板置于5%CO 2培养箱(37℃)中。
    16. 每2天更换培养基,直到收缩测定。

  3. 体外回缩测试
    1. 在微量离心管中混合10μgephrinA3-Fc/Fc和45μg抗人Fc抗体。
    2. 将管置于25℃的培养箱中。
    3. 1小时后,将预聚集的溶液放入器官型海马切片培养基中。 ephrinA3-Fc/Fc的最终浓度为10μg/ml
    4. 将板置于37℃的5%CO 2培养箱中
    5. 16小时后,用4%PFA的PBS溶液在室温下将海马切片固定在五彩纸屑片上30分钟。
    6. 用Vectashield固定介质将海马切片装载到载玻片上,并用盖玻片覆盖
  4. 成像和分析
    1. 图像树突棘在海马锥体神经元CA1区域使用共聚焦显微镜的初级/次级树突。 连续z-图像由具有0.1μm间隔的光学截面(1,024×1,024像素)组成,使用具有9x数字变焦的63x油浸物镜(数值孔径,1.3)。 图4显示了典型树突棘的z-堆积图像
    2. 重建z图像,并测量脊柱密度,脊柱长度,体积由Imaris长丝示踪剂。


      图4.树突棘回缩测定结果的实施例在用ephrinA3-Fc(右)或Fc(左)处理后来自海马切片的树突的区段。 比例尺:10μm。

食谱

  1. 培养基
    250ml含glutamax-1的MEM 125毫升马血清
    120ml含有3%D-葡萄糖的EBSS 5ml青霉素 - 链霉素 0.3ml制霉菌素
  2. 切割缓冲区
    487.5ml EBSS
    12.5 ml 1 M HEPES

致谢

该协议从Iwata等采用。 (2015)。 这项工作由JSPS KAKENHI(15H04263,16K14559)和MEXT KAKENHI(15H01454)支持。

参考文献

  1. Iwata,R.,Matsukawa,H.,Yasuda,K.,Mizuno,H.,Itohara,S。和Iwasato,T。(2015)。  发育RacGAPα2嵌合蛋白信号传导是成年树突棘形态学特征的决定因素。 J Neurosci 35(40):13728-13744。
  2. Mizuno,H.,Luo,W.,Tarusawa,E.,Saito,YM,Sato,T.,Yoshimura,Y.,Itohara,S。和Iwasato,T。(2014)。  NMDAR调节的层4神经元树突的动态在新生儿的脑皮层重组过程中。 em> Neuron 82(2):365-379。
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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. Iwata, R. and Iwasato, T. (2016). In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling. Bio-protocol 6(18): e1937. DOI: 10.21769/BioProtoc.1937.
  2. Iwata, R., Matsukawa, H., Yasuda, K., Mizuno, H., Itohara, S. and Iwasato, T. (2015). Developmental RacGAP α2-chimaerin signaling is a determinant of the morphological features of dendritic spines in adulthood. J Neurosci 35(40): 13728-13744.
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