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Neuroscience

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Published: June 19th, 2016

DOI:

10.3791/54096

1Anatomy and Neuroscience, Hamamatsu University School of Medicine, 2Cell and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT)

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance is the process by which newly formed neurons send axons to their target during development of the nervous system1,2. Developing axons carry a highly motile structure at their tip called the growth cone. The growth cone senses extracellular cues to navigate the axon's path. Guidance molecules, such as slit, semaphorin, and ephrins, can attract or repel axons depending on their interaction with suitable receptors and co-receptors on the axon1,3,4. The activated receptors transfer signals to the growth cone that affect its cytoskeletal organization for axon and growth-cone movements.

Various methods have....

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Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Hamamatsu University School of Medicine.

1. Preparation of Matrices

  1. Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up).
    Note: The following procedures should be performed under laminar flow.
  2. Blow compressed air or use transparent sticky tape to remove any dust from the striped side .......

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Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 h on stripes of fluorescently labeled control Fc (Figure 3A-C) or FLRT2-Fc (Figure 3D-F) alternating with non-labeled control Fc. In both cases, the neurons were aggregated and extended their axons as bundles. On the control Fc/Fc stripes, the neurons were distributed evenly on the fluorescently labeled and non-labeled stripes, and they extended their axons in random direction.......

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This protocol describes a stripe assay that uses recombinant protein and dissociated neurons from the E15.5 mouse hippocampus. This assay allows the efficient observation of repulsive, attractive, or neutral responses of neurons to a recombinant protein of interest placed in a striped pattern. A major advantage of this protocol is the simple method for generating the stripes, in which the protein is directly printed onto the surface of a plastic dish, compared to the traditional method, which requires special matrices, a.......

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This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

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Name Company Catalog Number Comments
15 mL centrifuge tube Violamo  1-3500-01
4% Paraformaldehyde (PFA) Nacalai 01954-85
Alexa Fluor 488 Goat anti-human IgG antibody Thermo Scientific A11013
Alexa Fluor 594 Donkey anti-mouse IgG antibody Thermo Scientific A-21203 Dilution 1/500
Anti-Tau1 antibody Chemicon MAB3420 Dilution 1/200
Antifade Thermo Scientific P7481 Alternative mounting media may be used
B27 supplement Thermo Scientific 17504-044 Dilution 1/50
Bovine serum albumin Sigma 01-2030-2
Cell strainer 100 um BD Falcon 352360
Centrifugation machine Kubota 2410
Cover glass 18mmx18mm Matsunami 18x18 mm No. 1
DAKO pen DAKO S2002 Alternative water-repellent pen may be used
Disposable scalpel Feather 2975#11
FBS Thermo Scientific 10437-028
Fluorecent microscope Nikon E600
Forceps No. 5 Fine Science Tools 11254-20
GlutaMAX Thermo Scientific 35050-061 Dilution 1/200
Hamilton Syringe Hamilton 805N 22 gauge, 50 uL
HBSS Thermo Scientific 14170-112
Human IgG, Fc Fragment Jackson 009-000-008
Laminin Thermo Scientific 23017-015
Neurobasal Thermo Scientific 21103-049
Normal Donkey Serum Jackson 017-000-121
PBS Nacalai 14249-24
Penicillin-Streptomycin Thermo Scientific 15070-063 Dilution 1/100
Plastic culture dish, 60 mm Thermo Scientific 150288
Silicone Matrices Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)
Stereo Microscope Olympus SZ61
Tip, 1000 uL Watson 125-1000S
Transparent sticky tape Tesa 57315 Alternative sticky tape may be used
Triton X-100 Sigma T8787
Trypan blue, 0.4% Bio-Rad 145-0013
Trypsin/EDTA Thermo Scientific 25300-054
Culture medium Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.

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