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Anterograde Axonal Tract Tracing
Publication Name:
Current Protocols in Neuroscience
Unit Number:
Unit 1.14
DOI:
10.1002/0471142301.ns0114s37
Online Posting Date:
November, 2006 Abstract
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
Materials
Basic Protocol: Anterograde Axonal Tract Tracing with PHA-L and BDA
Materials
- Rats (200 to 300 g)
- Ketamine×HCl
- Dormitor
- Iodine swabs
- 1% (v/v) hydrogen peroxide (H
2 O2 ) - Clay (e.g., soft children's play clay)
- PHA-L (see
- Post-operative analgesics (see appendix
- 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: Pressure Injection of BDA
Additional Materials (also see Basic Protocol )
- Rabbit anti-PHA-L antibody (Vector Labs)
- Appropriate secondary antibodies
- Microsyringe
- Additional reagents and equipment for rat perfusion fixation (unit
Looking for materials? Suppliers Guide
Figures
-
Figure 1.14.1Tract tracing setup. (A) Overall setup for tract tracing experiments. Note the stereotaxic table (A1), micromanipulator (A2), micropipet holder (A3), and current source (A4). (B) Backfilling of the micropipet. Note the oblique angle of the micropipet and the plastic tube attached to top of the micropipet. (C) Iontophoretic injection of tracer. Note the oblique injection and the attachment of the grounding electrode (black wire) to the skin of the rat. A thin silver wire connected to positive electrode is placed directly into the solution containing the tracer. -
Figure 1.14.2Routes of transport. Injection of BDA or PHA-L leads to anterograde transport (A). In some neuronal systems, the tracer may be retrogradely transported to the cell body and then anterogradely transported along axon collaterals (B). The retrograde and subsequent anterograde transport of tracer may lead to false-positive results. This bidirectional transport may be utilized to better examine the projections of axon collaterals of certain neuronal systems. -
Figure 1.14.3(A, B) Immunostaining for PHA-L shows a circumscribed injection site in the rat thalamus. (C) At the light microscopic level, labeling for the tracer shows a dense plexus of axonal fibers in the target region of the injection nucleus. (D) At the electron microscopic level, the tracer identifies an anterogradely labeled axon terminal (Te) forming an asymmetric synapse onto a dendrite (Den).
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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