Anterograde tracing

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In neuroscience, anterograde tracing is a research method that is used to trace axonal projections from their source (the cell body, or soma) to their point of termination (the synapse). A hallmark of anterograde tracing is the labeling of the presynaptic and the postsynaptic neuron(s). The crossing of the synaptic cleft is a vital difference between the anterograde tracers and the dye fillers used for morphological reconstruction. The complementary technique is retrograde tracing, which is used to trace neural connections from their termination to their source (i.e. synapse to cell body).[1] Both the anterograde and retrograde tracing techniques are based on the visualization of the biological process of axonal transport.

The anterograde and retrograde tracing techniques allow the detailed descriptions of neuronal projections from a single neuron or a defined population of neurons to their various targets throughout the nervous system.[2] These techniques allow the "mapping" of connections between neurons in a particular structure (e.g. the eye) and the target neurons in the brain. Much of what is currently known about connectional neuroanatomy was discovered through the use of the anterograde and retrograde tracing techniques.[1]


Several methods exist to trace projections originating from the soma towards their target areas. These techniques initially relied upon the direct physical injection of various visualizable tracer molecules (e.g. green fluorescent protein, lipophylic dyes or radioactively tagged amino acids) into the brain. These molecules are absorbed locally by the soma (cell body) of various neurons and transported to the axon terminals, or they are absorbed by axons and transported to the soma of the neuron. Other tracer molecules allow for the visualization of large networks of axonal projections extending from the neurons exposed to the tracer.[1]

Over the recent years viral vectors have been developed and implemented as anterograde tracers to identify the target regions of projecting neurons.[3][4]

Alternatively strategies are transsynaptic anterograde tracers, which can cross the synaptic cleft, labeling multiple neurons within a pathway. Those can also be genetic or molecular tracers.

Recently manganese-enhanced magnetic resonance imaging (MEMRI) has been used to trace functional circuits in living brains, as pioneered by Russ Jacobs,[5] Robia Paultler,[6] Alan Koretsky and Elaine Bearer.[7] The Mn2+ ion gives a hyperintense signal in T1-weighted MRI and thus serves as a contrast agent. Mn2+ enters through voltage dependent calcium channels, is taken into intracellular organelles and is transported by the endogenous neuronal transport system including kinesin-1, accumulating at distant locations.[8] Statistical parametric mapping of Mn accumulation in time-lapse images provides detailed information not only about neuronal circuitry but also about the dynamics of transport within them, and the location of distal connections.[9] This approach provides information about circuitry throughout the brain in living animals.

Genetic tracers[edit]

(see also Viral neuronal tracing)

In order to trace projections from a specific region or cell, a genetic construct, virus or protein can be locally injected, after which it is allowed to be transported anterogradely. Viral tracers can cross the synapse, and can be used to trace connectivity between brain regions across many synapses. Examples of viruses used for anterograde tracing are described by Kuypers.[10] Most well known are the herpes simplex virus type1 (HSV) and the rhabdoviruses.[10] HSV was used to trace the connections between the brain and the stomach, in order to examine the brain areas involved in viscero-sensory processing.[11] Another study used HSV type1 and type2 to investigate the optical pathway: by injecting the virus into the eye, the pathway from the retina into the brain was visualized.[12]

Viral tracers use a receptor on the host cell to attach to it and are then endocytosed. For example, HSV uses the nectin receptor and is then endocytosed. After endocytosis, the low pH inside the vesicle strips the envelope of the virion after which the virus is ready to be transported to the cell body. It was shown that pH and endocytosis are crucial for the HSV to infect a cell.[13] Transport of the viral particles along the axon was shown to depend on the microtubular cytoskeleton.[14]

Molecular tracers[edit]

There is also a group of tracers that consist of protein products that can be taken up by the cell and transported across the synapse into the next cell. Wheat-germ agglutinin (WGA) and Phaseolus vulgaris leucoagglutinin[15] are the most well known tracers, however they are not strict anterograde tracers: especially WGA is known to be transported anterogradely as well as retrogradely.[16] WGA enters the cell by binding to oligosaccharides, and is then taken up via endocytosis via a caveolae-dependent pathway.[17][18]

Other anterograde tracers widely used in neuroanatomy are the biotinylated dextran amines (BDA), also used in retrograde tracing.

Partial list of studies using this technique[edit]

The anterograde tracing technique is now a widespread research technique. The following are a partial list of studies that have used anterograde tracing techniques:

  • Talay, M., Richman, E. B., Snell, N. J., Hartmann, G. G., Fisher, J. D., Sorkaç, A., Santoyo, J. F., Chou-Freed, C., Nair, N., Johnson, M., Szymanski, J. R., & Barnea, G. (November 2017). Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango. Neuron, 96(4), 783–795.e4.
  • Deller T, Naumann T, Frotscher M (November 2000). "Retrograde and anterograde tracing combined with transmitter identification and electron microscopy". Journal of Neuroscience Methods. 103 (1): 117–26. doi:10.1016/S0165-0270(00)00301-0. PMID 11074101. S2CID 13534170.
  • Kressel M (April 1998). "Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry". The Journal of Histochemistry and Cytochemistry. 46 (4): 527–33. doi:10.1177/002215549804600413. PMID 9575040. S2CID 17642689. Archived from the original on 2013-04-15.
  • Haberl MG, Viana da Silva S, Guest JM, Ginger M, Ghanem A, Mulle C, Oberlaender M, Conzelmann KK, Frick A (Apr 2014). "An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology". Brain Structure & Function. 220 (3): 1369–79. doi:10.1007/s00429-014-0730-z. PMC 4409643. PMID 24723034.
  • Chamberlin NL, Du B, de Lacalle S, Saper CB (May 1998). "Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS". Brain Research. 793 (1–2): 169–75. doi:10.1016/s0006-8993(98)00169-3. PMC 4961038. PMID 9630611.
  • Luppi PH, Fort P, Jouvet M (November 1990). "Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons". Brain Research. 534 (1–2): 209–24. doi:10.1016/0006-8993(90)90131-T. PMID 1705851. S2CID 25326054.

See also[edit]


  1. ^ a b c Dale Purves; George J. Augustine; David Fitzpatrick; William C. Hall; Anthony-Samuel Lamantia; James O. Mcnamara; Leonard E. White, eds. (2008). Neuroscience (4th ed.). Sunderland, Massachusetts: Sinauer. pp. 16–18 (of 857 total). ISBN 978-0-87893-697-7.
  2. ^ Lanciego, Jose L.; Wouterlood, Floris G. (1 May 2020). "Neuroanatomical tract-tracing techniques that did go viral". Brain Structure and Function. pp. 1193–1224. doi:10.1007/s00429-020-02041-6. Retrieved 2 February 2024.
  3. ^ Oh SW, Harris JA, Ng L, Winslow B, Cain N, Mihalas S, et al. (April 2014). "A mesoscale connectome of the mouse brain". Nature. 508 (7495): 207–14. Bibcode:2014Natur.508..207O. doi:10.1038/nature13186. PMC 5102064. PMID 24695228.
  4. ^ Haberl MG, Viana da Silva S, Guest JM, Ginger M, Ghanem A, Mulle C, Oberlaender M, Conzelmann KK, Frick A (Apr 2014). "An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology". Brain Structure & Function. 220 (3): 1369–79. doi:10.1007/s00429-014-0730-z. PMC 4409643. PMID 24723034.
  5. ^ Pautler RG, Mongeau R, Jacobs RE (July 2003). "In vivo trans-synaptic tract tracing from the murine striatum and amygdala utilizing manganese enhanced MRI (MEMRI)". Magnetic Resonance in Medicine. 50 (1): 33–9. doi:10.1002/mrm.10498. PMID 12815676.
  6. ^ Pautler RG, Silva AC, Koretsky AP (November 1998). "In vivo neuronal tract tracing using manganese-enhanced magnetic resonance imaging". Magnetic Resonance in Medicine. 40 (5): 740–8. doi:10.1002/mrm.1910400515. PMID 9797158. S2CID 13996533.
  7. ^ Bearer EL, Falzone TL, Zhang X, Biris O, Rasin A, Jacobs RE (2007). "Role of neuronal activity and kinesin on tract tracing by manganese-enhanced MRI (MEMRI)". NeuroImage. 37 (Suppl 1): S37–46. doi:10.1016/j.neuroimage.2007.04.053. PMC 2096707. PMID 17600729.
  8. ^ Medina CS, Biris O, Falzone TL, Zhang X, Zimmerman AJ, Bearer EL (January 2017). "2+ is impaired by deletion of KLC1, a subunit of the conventional kinesin microtubule-based motor". NeuroImage. 145 (Pt A): 44–57. doi:10.1016/j.neuroimage.2016.09.035. PMC 5457905. PMID 27751944.
  9. ^ Bearer EL, Manifold-Wheeler BC, Medina CS, Gonzales AG, Chaves FL, Jacobs RE (October 2018). "Alterations of functional circuitry in aging brain and the impact of mutated APP expression". Neurobiology of Aging. 70: 276–290. doi:10.1016/j.neurobiolaging.2018.06.018. PMC 6159914. PMID 30055413.
  10. ^ a b Kuypers HG, Ugolini G (February 1990). "Viruses as transneuronal tracers". Trends in Neurosciences. 13 (2): 71–5. doi:10.1016/0166-2236(90)90071-H. PMID 1690933. S2CID 27938628.
  11. ^ Rinaman L, Schwartz G (March 2004). "Anterograde transneuronal viral tracing of central viscerosensory pathways in rats". The Journal of Neuroscience. 24 (11): 2782–6. doi:10.1523/JNEUROSCI.5329-03.2004. PMC 6729508. PMID 15028771.
  12. ^ Norgren RB, McLean JH, Bubel HC, Wander A, Bernstein DI, Lehman MN (March 1992). "Anterograde transport of HSV-1 and HSV-2 in the visual system". Brain Research Bulletin. 28 (3): 393–9. doi:10.1016/0361-9230(92)90038-Y. PMID 1317240. S2CID 4701001.
  13. ^ Nicola AV, McEvoy AM, Straus SE (May 2003). "Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells". Journal of Virology. 77 (9): 5324–32. doi:10.1128/JVI.77.9.5324-5332.2003. PMC 153978. PMID 12692234.
  14. ^ Kristensson K, Lycke E, Röyttä M, Svennerholm B, Vahlne A (September 1986). "Neuritic transport of herpes simplex virus in rat sensory neurons in vitro. Effects of substances interacting with microtubular function and axonal flow [nocodazole, taxol and erythro-9-3-(2-hydroxynonyl)adenine]". The Journal of General Virology. 67 ( Pt 9) (9): 2023–8. doi:10.1099/0022-1317-67-9-2023. PMID 2427647.
  15. ^ Smith Y, Hazrati LN, Parent A (April 1990). "Efferent projections of the subthalamic nucleus in the squirrel monkey as studied by the PHA-L anterograde tracing method". The Journal of Comparative Neurology. 294 (2): 306–23. doi:10.1002/cne.902940213. PMID 2332533. S2CID 9667393.
  16. ^ Damak S, Mosinger B, Margolskee RF (October 2008). "Transsynaptic transport of wheat germ agglutinin expressed in a subset of type II taste cells of transgenic mice". BMC Neuroscience. 9: 96. doi:10.1186/1471-2202-9-96. PMC 2571104. PMID 18831764.
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  18. ^ Gao X, Wang T, Wu B, Chen J, Chen J, Yue Y, Dai N, Chen H, Jiang X (December 2008). "Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles". Biochemical and Biophysical Research Communications. 377 (1): 35–40. doi:10.1016/j.bbrc.2008.09.077. PMID 18823949.