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Retrograde Axonal Tracing with Fluorescent Markers

Brett R. Schofield1

1Northeastern Ohio Universities College of Medicine, Rootstown, Ohio

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
Unit 1.17
DOI: 
10.1002/0471142301.ns0117s43
Online Posting Date: 
April, 2008
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Abstract

The growth of fluorescence imaging technology and the development of sensitive fluorescent retrograde tracers has provided many new approaches for analyzing neuronal circuits. Fluorescent markers provide unparalleled opportunity for combining axonal tract tracing with techniques such as immunohistochemistry or physiological recording. This unit describes the use of six different fluorescent tracers: Fast Blue, fluorescein dextran, FluoroGold, FluoroRuby, red beads, and green beads. Guidance is provided on how to choose a tracer for a particular experiment, and three methods are described for injecting the tracers, including pressure injection through a microsyringe or a micropipet, and iontophoretic injection through a micropipet. Criteria for selecting the most appropriate method are discussed. The protocols provide the information necessary to take advantage of the numerous fluorescent tracers that are available and to apply them to a wide variety of scientific questions. Curr. Protoc. Neurosci. 43:1.17.1-1.17.24. © 2008 by John Wiley & Sons, Inc.

Keywords: neuroanatomy; tract-tracing; axonal transport; circuit

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Pressure Injection of Tracer Through a Micropipet Attached to a Nanoliter Injector
  • Alternate Protocol 1: Iontophoretic Injection through a Micropipet using a Current Source
  • Alternate Protocol 2: Pressure Injection through Microsyringe
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Pressure Injection of Tracer Through a Micropipet Attached to a Nanoliter Injector

 Materials
  • Tracers: (see Table 1.17.2 for recipes and suppliers)
     
    Table 1.17.2 Tracer Solutions
    TracerProduct nameSourceSolution
    Fast BlueFast BlueEMS-Chemie GmbH5% in water
    fluorescein dextran (FluoroEmerald)dextran, fluorescein, 10,000 mol. wt., anionic Catalog Number D-1821Invitrogen http://www.probes.invitrogen.com10% in saline
    FluoroGoldFluoroGoldFluorochrome LLC http://www.fluorochrome.com4% in water (for pressure) 2% in saline (for iontophoresis)
    FluoroRubyDextran, tetramethylrhodamine, 10,000 mol. wt., lysine fixable (Fluoro-Ruby) Catalog Number D-1817Invitrogen, see above10% in saline
    green beadsgreen RetroBeadsLumaFluor, Inc. http://www.lumafluor.comShipped in solution; use undiluted
    red beadsred RetroBeadsLumaFluor, Inc. http://www.lumafluor.comShipped in solution; use undiluted
    • Fast Blue
    • FluoroRuby
    • Fluorescein dextran
    • FluoroGold
    • Green RetroBeads
    • Red RetroBeads
  • Mineral oil
  • Animals
  • Atropine
  • General anesthetic (see appendix 4B)
  • 70% ethanol
  • Iodine/povidone solution (e.g., Betadine, available at most drug stores)
  • Eye ointment (Moisture Eyes by Bausch and Lomb or equivalent; available at most drug stores)
  • Bupivacaine (long-lasting local anesthetic)
  • Sterile saline
  • Squirt bottle with deionized water
  • Ketoprofen (long-lasting post-operative analgesic)
  • 4% paraformaldehyde solution (see recipe)
  • Fixative (e.g., 4% paraformaldehyde) with 30% sucrose (50 ml)
  • 0.1 M phosphate buffer, pH 7.4 (see recipe)
  • Thionin, optional
  • DPX neutral mounting medium (Aldrich, cat. no. 317616)
  • Ultracentrifuge tubes
  • Glass-bead sterilizer
  • Nanoliter Injector (Nanoliter 2000, World Precision Instruments)
  • Stereotaxic frame with manipulators, electrode holders
  • Micropipet puller (Model 720, David Kopf Instruments)
  • Capillary tubes designed for use with Nanoliter Injector
  • Electrode puller
  • 1-ml tuberculin syringes
  • Upright microscope (to measure micropipet tip)
  • Hand-held lens (2× – 4×)
  • Heating blanket with temperature probe
  • Scale
  • Surgical tools (Fig. 1.17.1A; Bernsco Surgical Supply; Henry Schein)
    • Surgical clipper
    • Scalpel with no. 10 blade
    • Retractor
    • Elevator
    • Dental drill with bit
    • Excavator
    • Towel clamps
    • Small rongeurs
    • Suture tool (needle holder and scissors)
    • 30-G needle mounted on 1-ml syringe barrel
    • Fine forceps (blunt tip)
    • Tissue forceps
  • Sterile 2×2–in. sponges
  • Lab jack
  • Fabric tape
  • Sterile drape
  • Surgical lamp/fiber optic light source
  • Surgical microscope
  • Surgical marker
  • 10-µl Hamilton microsyringe (one for each tracer to be used)
  • Glassine (weighing paper)
  • Small disposable beakers
  • Sterile cotton-tipped applicators
  • Sterile Gelfoam (Harvard Apparatus)
  • 3-0 suture, 18” silk with FS-3 needle
  • Mini ice cube trays (http://homebarsupplys.com)
  • Petri dishes
  • no. 1 ½ coverslips, 22 × 50–mm
  • Aluminum foil
  • Black slide boxes (25 slides/box; e.g., Thomas Scientific, cat. no. 6708L32)
  • Single-edged razor blade
  • Fluorescence microscope with appropriate light source and filters
  • Camera system for documentation of data
  • Additional reagents and equipment for anesthetizing the animal (appendix 4B), perfusion fixation (unit 1.1), sectioning frozen brain with sliding microtome (unit 1.1, Basic Protocol 4), mounting sections on gelatin-coated slides (unit 1.1, Basic Protocol 4), and thionin stain (unit 1.1 Basic Protocol 7)

Alternate Protocol 1: Iontophoretic Injection through a Micropipet using a Current Source

 Additional Materials (also see Basic Protocol)
  • Capillary tubes with microfilament (to make micropipets; World Precision Instruments, no. 1B150F-4)
  • Midgard Precision current source (Stoelting Co., no. 51595)

Alternate Protocol 2: Pressure Injection through Microsyringe

 Additional Materials (also see Basic Protocol)
  • 1-µl microsyringe (one for each tracer to be used)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 1.17.1
    Surgical equipment. (A) Note stereotaxic frame, lab jack with heating pad on top, temperature probe for heating pad and blanket control unit; surgical microscope, fabric tape and instruments. (B) Nanoliter Injector mounted on a stereotaxic manipulator on a centering stand. A micropipet (arrow) is mounted on the Injector. Also shown are the Nanoliter control unit, a small lens for visually aligning the micropipet tip with the centering stand, and allen wrenches for adjusting the manipulator. During surgery the lab jack and heating blanket are covered by a sterile drape.

    View Image
  • Figure 1.17.2
    (A) Iontophoresis equipment, including current source, negative lead (to be clipped to animal's skin) and positive lead going to a micropipet (arrow). The micropipet is mounted on a micromanipulator on a centering stand. (B) Close-up of micropipet (arrow) clamped in the electrode holder, with positive electrical lead inserted into back of micropipet (arrowhead). (C). Setup for pressure injection with a 1-µl Hamilton microsyringe. The microsyringe is mounted on an electrode holder (on a stereotaxic manipulator) with a clamp and fabric tape so that the needle protrudes with sufficient clearance for entry into the brain.

    View Image
  • Figure 1.17.3
    Typical injection sites. Each panel shows the center of an injection of tracer into the left inferior colliculus of a guinea pig. (A) Large injection of FluoroGold (FG) made with a microsyringe (Alternate Protocol 2). Note central area of necrosis. (B) Medium size injection of FluoroGold made with Nanoliter Injector (Basic Protocol). (C, D) Injections of green beads (GB) made with a microsyringe (C) or Nanoliter Injector (D). Note the irregular outlines of the injection sites. (E) Small injection of Fast Blue (FB) made with a microsyringe. (F). Small injection of FluoroRuby (FR) made with a microsyringe. Note diffuse, poorly-defined “edge” of the injection site. Transverse sections; scale bar = 1 mm..

    View Image
  • Figure 1.17.4
    Typical appearance of labeled cells. All cells are in auditory cortex and were labeled by injections of tracer into the brainstem in guinea pigs. The cells were labeled with FluoroGold (A), Fast Blue (B), red beads (C), green beads (D), FluoroRuby (E), or fluorescein dextran (F). Abbreviations: FB, Fast Blue; FD, fluorescein dextran; FG, FluoroGold; FR, FluoroRuby; GB, green beads; RB, red beads. Scale bar = 20 µm..

    View Image

Literature Cited

Literature Cited
    Akintunde, A. and Buxton, D.F. 1992. Quadruple labeling of brain-stem neurons: A multiple retrograde fluorescent tracer study of axonal collateralization. J. Neurosci. Meth. 45: 15-22.
    Clancy, B. and Cauller, L.J. 1998. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. J. Neurosci. Meth. 83: 97-102.
    Katz, L.C., Burkhalter, A. and Dreyer, W.J. 1984. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310: 498-500.
    Köbbert, C., Apps, R., Bechmann, I., Lanciego, J.L., Mey, J., and Thanos, S. 2000. Current concepts in neuroanatomical tracing. Prog. Neurobiol. 62: 327-351.
    Kuypers, H.G.J.M. and Huisman, A.M. 1984. Fluorescent neuronal tracers. In Advances in Cellular Neurobiology, vol.5. (S. Federoff, ed.) pp. 307-340. Academic Press, N.Y.
    Lanciego, J.L., Wouterlood, F.G., Erro, E., Arribas, J., Gonzalo, N., Urra, X., Cervantes, S., and Gimenez-Amaya, J.M. 2000. Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section. J. Neurosci. Meth. 103: 127-135.
    Novikova, L., Novikov, L., and Kellerth, J.O. 1997. Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates. J. Neurosci. Meth. 74: 9-15.
    Schofield, B.R. and Cant, N.B. 1999. Descending auditory pathways: Projections from the inferior colliculus contact superior olivary cells that project bilaterally to the cochlear nuclei. J. Comp. Neurol. 409: 210-223.
    Schofield, B.R., Coomes, D.L., and Schofield, R.M. 2006. Cells in auditory cortex that project to the cochlear nucleus in guinea pigs. J. Assoc. Res. Otolaryngol. 7: 95-109.
    Schofield, B.R., Schofield, R.M., Sorensen, K.A., and Motts, S.D. 2007. On the use of retrograde tracers for identification of axon collaterals with multiple fluorescent retrograde tracers. Neuroscience 146: 773-783.
     
 
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