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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

Ryohei Iwata Ryohei Iwata
Takuji Iwasato Takuji Iwasato

发布: 2016年09月20日第6卷第18期 DOI: 10.21769/BioProtoc.1937 浏览次数: 10300

评审: Soyun KimShai BerlinEmmanuelle Berret

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Oct 2015

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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) search Retraction assay (收缩试验) search EphA (EphA) search Spinogenesis (spinogenesis) search

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

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 细胞机理

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