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In neuroscience, anterograde tracing is a research method which is used to trace axonal projections from their source (the cell body or soma) to their point of termination (the synapse). 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). 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 population of neurons to their various targets throughout the nervous system. 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.
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.
The aforementioned tracers such as GFP and DiI are technically not anterograde tracers: they do not selectively travel in a certain direction and are not actively transported by the cell. Furthermore, they do not cross the synaptic cleft, and therefore only label the neurons that were directly at the site of tracer application. These dyes are mostly used as a filler to mark cells that have been recorded using electrophysiology: this allows the cells that have been recorded to also be morphologically reconstructed in 3D.
The anterograde tracers that can cross the synaptic cleft and label multiple neurons within a pathway can be divided in two categories: genetic and molecular tracers.
(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. Most well known are the Herpes simplex virus type1 (HSV) and the Rhabdoviruses. 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. 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.
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. Transport of the viral particles along the axon was shown to depend on the microtubular cytoskeleton.
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 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. WGA enters the cell by binding to Oligosaccharides,and is then taken up via endocytosis via a caveolae-dependent pathway.
Partial list of studies using this techniqueEdit
The anterograde tracing technique is now a widespread research technique. The following are a partial list of studies that have used anterograde tracing techniques:
- 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.
- 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.
- 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.
- ↑ 1.0 1.1 1.2 (2008) Dale Purves, George J. Augustine, David Fitzpatrick, William C. Hall, Anthony-Samuel Lamantia, James O. Mcnamara, Leonard E. White Neuroscience, 4th, 16–18 (of 857 total), Sunderland, Massachusetts: Sinauer.
- ↑ Murphy MC, Fox EA (July 2007). Anterograde Tracing Method using DiI to Label Vagal Innervation of the Embryonic and Early Postnatal Mouse Gastrointestinal Tract. Journal of Neuroscience Methods 163 (2): 213–25.
- ↑ Vuksic M, Del Turco D, Bas Orth C, et al. (2008). 3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse. Hippocampus 18 (4): 364–75.
- ↑ 4.0 4.1 Kuypers HG, Ugolini G (February 1990). Viruses as transneuronal tracers. Trends in Neurosciences 13 (2): 71–5.
- ↑ Rinaman L, Schwartz G (March 2004). Anterograde transneuronal viral tracing of central viscerosensory pathways in rats. The Journal of Neuroscience 24 (11): 2782–6.
- ↑ 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.
- ↑ 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.
- ↑ 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 (9): 2023–8.
- ↑ 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.
- ↑ Damak S, Mosinger B, Margolskee RF (2008). Transsynaptic transport of wheat germ agglutinin expressed in a subset of type II taste cells of transgenic mice. BMC Neuroscience 9: 96.
- ↑ Broadwell RD, Balin BJ (December 1985). Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo. The Journal of Comparative Neurology 242 (4): 632–50.
- ↑ Gao X, Wang T, Wu B, et al. (December 2008). Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles. Biochemical and Biophysical Research Communications 377 (1): 35–40.
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