Anterograde Axonal Tract Tracing

Dinesh V. Raju1, Yoland Smith1

1 Yerkes National Primate Research Center, Emory University, Atlanta
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.14
DOI:  10.1002/0471142301.ns0114s37
Online Posting Date:  November, 2006
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The mammalian brain contains a myriad of interconnected regions. An examination of the complex circuitry of these areas requires sensitive neuroanatomical tract tracing techniques. The anterograde tracers, Phaseolus vulgaris leucoagglutinin (PHA‐L) and biotinylated dextran amines (BDA) are powerful tools that can be used to label fiber tracts that project from one particular brain region. When injected iontophoretically, PHA‐L and BDA are readily taken up by neurons and transported anterogradely along their axonal tracts. Combined with immunocytochemistry for neurotransmitters, neuropeptides, and receptors, tract tracing methods may be used to elucidate the phenotype of synapses that form the microcircuitry of specific neural systems.

Keywords: Phaseolus vulgaris leucoagglutinin; dextran amines; tracer; light microscopy; electron microscopy; neuroanatomical connectivity

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Table of Contents

  • Basic Protocol 1: Anterograde Axonal Tract Tracing with PHA‐L and BDA
  • Alternate Protocol 1: Pressure Injection of BDA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Anterograde Axonal Tract Tracing with PHA‐L and BDA

  • Rats (200 to 300 g)
  • Ketamine⋅HCl
  • Dormitor
  • Iodine swabs
  • 1% (v/v) hydrogen peroxide (H 2O 2)
  • Clay (e.g., soft children's play clay)
  • PHA‐L (see recipe) or BDA (10 kD; see recipe)
  • Post‐operative analgesics (see appendix 4B)
  • Stereotaxic frame with micromanipulator and electrode holder (David Knopf)
  • Glass capillaries (1.0‐mm o.d., 0.5‐mm i.d.)
  • Electrode puller
  • Rat housing
  • Electric clipper
  • Ear bars
  • Scalpel
  • Retractors
  • Gauze or cotton‐tipped applicators, sterile
  • Magnifying glass (2× with 5× to 10× monocular)
  • Drill and bit
  • 0.2‐ml microcentrifuge tubes
  • Small plastic tubing (1‐mm diameter)
  • 3‐ and 5‐ml syringe
  • Needles (any gauge), 1 in.
  • Silver wire (0.003 in. diameter)
  • Current source device (Stoelting)
  • Sutures or skin staples

Alternate Protocol 1: Pressure Injection of BDA

  • Rabbit anti‐PHA‐L antibody (Vector Labs)
  • Appropriate secondary antibodies
  • Microsyringe
  • Additional reagents and equipment for rat perfusion fixation (unit 1.1) and tissue processing (unit 1.2)
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Literature Cited

Literature Cited
   Brandt, H.M. and Apkarian, A.V. 1992. Biotin‐dextran: A sensitive anterograde tracer for neuroanatomic studies in rat and monkey. J. Neurosci. Methods 45:35‐40.
   Chen, S. and Aston‐Jones, G. 1998. Axonal collateral‐collateral transport of tract tracers in brain neurons: False anterograde labelling and useful tool. Neuroscience 82:1151‐1163.
   Cliffer, K.D. and Giesler, G.J. Jr. 1988. PHA‐L can be transported anterogradely through fibers of passage. Brain Res. 458:185‐191.
   Fritzsch, B. 1993. Fast axonal diffusion of 3000 molecular weight dextran amines. J. Neurosci. Methods 50:95‐103.
   Gerfen, C. and Sawchenko, P. 1984. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA‐L). Brain Res. 290:219‐238.
   Gerfen, C.R., Sawchenko, P.E., and Carlsen, J. 1989. The PHA‐L anterograde axonal tracing method. In Neuroanatomical Tract‐Tracing Methods 2, Recent Progress (L. Heimer and L.Z. Zaborszky, eds.) pp. 19‐47. Plenum Press, New York.
   Groenewegen, H.J. and Wouterlood, F.G. 1990. Light and electron microscopic tracing of neuronal connections with Phaseolus vulgaris‐leucoagglutinin (PHA‐L), and combination with other neuroanatomical techniques. In Handbook of Chemical Neuroanatomy, Vol. 8: Analysis of Neuronal Microcircuits and Synaptic Interactions (A. Bjorklund, T. Hokfelt, F.G. Wouterlood, and A.N. van den Pol, eds.) pp. 47‐124. Elsevier Science, Amsterdam.
   Lei, W., Jiao, Y., Del Mar, N., and Reiner, A. 2004. Evidence for differential cortical input to direct pathway versus indirect pathway striatal projection neurons in rats. J. Neurosci. 24:8289‐8299.
   McFarland, N.R. and Haber, S.N. 2001. Organization of thalamostriatal terminals from the ventral motor nuclei in the macaque. J. Comp. Neurol. 429:321‐336.
   Medina, L. and Reiner, A. 1997. The efferent projections of the dorsal and ventral pallidal parts of the pigeon basal ganglia, studied with biotinylated dextran amine. Neuroscience 81:773‐802.
   Pare, D. and Smith, Y. 1996. Thalamic collaterals of corticostriatal axons: Their termination field and synaptic targets in cats. J. Comp. Neurol. 372:551‐567.
   Rajakumar, N., Elisevich, K., and Flumerfelt, B.A. 1993. Biotinylated dextran: A versatile anterograde and retrograde neuronal tracer. Brain Res. 607:47‐53.
   Reiner, A., Veenman, C.L., Medina, L., Jiao, Y., Del Mar, N., and Honig, M.G. 2000. Pathway tracing using biotinylated dextran amines. J. Neurosci. Methods 103:23‐37.
   Schofield, B.R. 1990. Uptake of Phaseolus vulgaris leucoagglutinin (PHA‐L) by axons of passage. J. Neurosci. Methods 35:47‐56.
   Shink, E., Bevan, M.D., Bolam, J.P., and Smith, Y. 1996. The subthalamic nucleus and the external pallidum: Two tightly interconnected structures that control the output of the basal ganglia in the monkey. Neuroscience 73:335‐357.
   Sidibe, M. and Smith, Y. 1996. Differential synaptic innervation of striatofugal neurones projecting to the internal or external segments of the globus pallidus by thalamic afferents in the squirrel monkey. J. Comp. Neurol. 365:445‐465.
   Smith, Y. 1992. Anterograde tracing with PHA‐L and biocytin at the electron microscopic level. In Experimental Neuroanatomy, The Practical Approach Series (J.P. Bolam, ed.) pp. 61‐79. Oxford University Press, New York.
   Smith, Y., Bevan, M.D., Shink, E., and Bolam, J.P. 1998. Microcircuitry of the direct and indirect pathways of the basal ganglia. Neuroscience 86:353‐387.
   Veenman, C.L., Reiner, A., and Honig, M.G. 1992. Biotinylated dextran amine as an anterograde tracer for single‐ and double‐labeling studies. J. Neurosci. Methods 41:239‐254.
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