Time‐Lapse Imaging of Glial–Axonal Interactions

Kalliopi Ioannidou1, Julia M. Edgar1, Susan C. Barnett2

1 These authors contributed equally to this work, 2 Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.23
DOI:  10.1002/0471142301.ns0223s72
Online Posting Date:  July, 2015
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In the central nervous system (CNS), myelin is formed by oligodendrocytes that are derived from precursor cells, known as oligodendrocyte precursor cells (OPCs). Successive stages of OPC interactions with the axons can be visualized in vitro and ex vivo using mixed neural cell cultures and pieces of intact spinal cord, respectively. OPCs and their differentiation can be imaged using cell‐type‐specific markers or green fluorescent protein (GFP) tags. This protocol describes methodology for generating these two systems for time‐lapse imaging of dynamic cell interactions using fluorescent and 2‐photon microscopy. © 2015 by John Wiley & Sons, Inc.

Keywords: myelination; CNS; time‐lapse imaging

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Myelinating Cultures
  • Basic Protocol 2: Spinal Cord Transplantation and OPC Visualization Ex Vivo
  • Support Protocol 1: Generation of Neurospheres to Add to Myelinating Cultures or to Transplant into Shiverer Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Myelinating Cultures

  • C57BL/6 mice pregnant with E13.5 embryos (day of plug is E0)
  • 70% (v/v) ethanol
  • L15 medium (Life Technologies, cat. no. 11415‐049)
  • HBSS (Hanks balanced salt solution, without Ca2+ and Mg2+; Life Technologies, cat. no. 14170‐088)
  • 2.5% (w/v) trypsin (Life Technologies, cat. no. 15090‐046)
  • Soybean trypsin inhibitor and DNase (SD; see recipe)
  • Plating medium (PM; see recipe)
  • 4% (w/v) trypan blue in PBS
  • Poly‐L‐lysine (PLL; see recipe)
  • Differentiation medium (DM; see recipe)
  • CO 2
  • Induction box for euthanasia
  • Dissecting microscope
  • 15‐ml conical tube (Falcon)
  • Bench top centrifuge
  • Scalpel blade, no. 10 (Swann)
  • Surgical tools (World Precision Instruments), including large pair dissecting scissors, small pair dissecting scissors, blunt forceps, two sets fine forceps (Dumont #5)
  • 13‐mm coverslips, thickness no. 1 (use only VWR International, cat. no. 631‐0149), autoclaved and coated with PLL
  • 35‐mm and 100‐mm Petri dishes (Sterilin)
  • 35‐mm glass‐bottom microwell Petri dishes, with 14‐mm diameter microwell coated with PLL (MakTek Corp., cat. no. P35G‐1.5‐14‐C) (the time‐lapse microscope perfect focus system (PFS) only functions with glass‐bottom dishes)
  • Wet ice
  • Bijou containers (e.g., SterilinTM 7 ml polystyrene; Thermo Scientific, cat. no. 129A)
  • Glass pipets (Thermo Scientific)
  • Hemacytometer
  • Epifluorescence time‐lapse microscope (e.g., Nikon TE2000 fitted with a Nikon perfect focus system)
  • Additional reagents and equipment for mouse euthanasia ( appendix 4H; Donovan and Brown, ) and counting cells with a hemocytometer and trypan blue staining ( appendix 3B; Phelan, )
NOTE: The following is a general protocol for making myelinating co‐cultures (Sørensen et al., ; Thomson et al., ). To specifically visualize glial–axonal interactions and myelination, it is best to add neurospheres labeled with cytosolic or membrane‐tagged GFP on days 12 to 18. This can be done by generating neurospheres ( protocol 2) from transgenic mice or by exogenously labeling neurospheres by infection with viruses carrying farnesylated GFP (farns‐GFP), which targets cell membranes (Ioannidou et al., ). Neurospheres can be added at any time point depending on the scientific question. In general, differentiation of OPCs begins day 12 when medium is switched to DM minus insulin. Alternatively, generate the cultures from mice harboring fluorescently labeled oligodendroglia.

Basic Protocol 2: Spinal Cord Transplantation and OPC Visualization Ex Vivo

  • Recipient P21 shiverer mice
  • Isoflurane
  • Neurosphere cell suspension (∼5 × 104 cells/μl; protocol 3Support Protocol)
  • Betadine skin wipes
  • Pre‐warmed neurobasal A medium (Life Technologies, cat. no. 10888‐022), bubbled with 95% O 2 and 5% CO 2 for at least 15 min before imaging
  • Vetbond tissue adhesive (3M)
  • Operating and dissecting microscopes
  • Analgesic (e.g., Vetergesic, Ceva Animal Health)
  • Heating pad
  • Electric shaver
  • O 2 supply and nose mask
  • Induction box for anesthesia
  • Pulled‐glass electrodes to inject cells
  • CellTram oil injection system (Eppendorf) and micromanipulator
  • Very fine forceps (Dumont #5), two sets
  • Scissors, absorbable sutures, and suture holder to close wound
  • Dissecting board
  • Wet ice
  • Blunt forceps (Dumont #5)
  • Scalpel blade and holder
  • Pecon POC‐R2 cell cultivation system
  • 2‐photon imaging system
    • LaVision BioTec 2‐photon TRIM scope with Nikon Eclipse TE2000 inverted stand and Olympus long working‐distance 20×‐0.95 NA water immersion objective, or a Zeiss 7MP system with Axio Imager upright stand and 20×‐1.0 NA water immersion objective
    • Coherent Chameleon II laser tuned to 830 nm
    • Non‐descanned detectors (NDD, Hamamatsu H6780‐01‐LV 1 M for <500 nm detection and H6780‐20‐LV 1 M for >500 nm detection)
  • Additional reagents and equipment for mouse anesthesia and analgesia ( appendix 4B; Davis, ), euthanasia ( appendix 4H; Donovan and Brown, ), optimization of laser sources (McConnell, ), and 2‐photon microscopy (unit 2.9; Belluscio, ; Benninger and Piston, )

Support Protocol 1: Generation of Neurospheres to Add to Myelinating Cultures or to Transplant into Shiverer Mice

  • 70% Ethanol
  • P1 mouse pups, C57BL/6 or C57BL/6‐Tg(ACTB‐EGFP)1Osb/J (Jackson Labs)
  • Sodium pentobarbital (e.g., 200 mg/ml Euthatal, 0.03 ml per pup; Henry Schein Medical)
  • L15 medium (Life Technologies, cat. no. 11415‐049), with antibiotics (5000 U/ml penicillin‐streptomycin; Life Technologies, cat. no. 15070‐063)
  • Neurosphere medium (see recipe)
  • 100 μg/ml EGF (Peprotech, cat. no. 315‐09)
  • Scalpel blade, no. 10 (Swann)
  • Dissecting microscope
  • 35‐mm Petri dishes
  • Wet ice
  • Small pair of dissecting scissors
  • Curved‐tip forceps (Dumont #7; World Precision Instruments)
  • Bijou containers
  • Glass pipets, flame‐polished (Thermo)
  • T75 tissue culture flasks (Nunc)
  • Additional reagents and equipment for mouse euthanasia ( appendix 4H; Donovan and Brown, )
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Literature Cited

Literature Cited
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  Ben Geren, B. 1954. The formation from the Schwann cell surface of myelin in the peripheral nerves of chick embryos. Exp. Cell Res. 7:558‐562.
  Benninger, R. K. and Piston, D. W. 2013. Two‐photon excitation microscopy for the study of living cells and tissues. Curr. Protoc. Cell. Biol. 59:4.11.1‐4.11.24.
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  Ioannidou, K., Anderson, K.I., Strachan, D., Edgar, J.E., and Barnett, S.C. 2012. Time‐lapse imaging of the dynamics of CNS glial–axonal interactions in vitro and ex vivo. Plos One 7:e30775.
  Ioannidou, K., Anderson, K.I., Strachan, D., Edgar, J.M., and Barnett, S.C. 2014. Astroglial–axonal interactions during early stages of myelination in mixed cultures using in vitro and ex vivo imaging techniques. BMC Neurosci. 15:59.1‐59.12.
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