Retrograde tracing

Retrograde tracing is a research method used in neuroscience to trace neural connections from their point of termination (the synapse) to their source (the cell body). Retrograde tracing techniques allow for detailed assessment of neuronal connections between a target population of neurons and their inputs 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. The opposite technique is anterograde tracing, which is used to trace neural connections from their source to their point of termination (i.e. from cell body to synapse). Both the anterograde and retrograde tracing techniques are based on the visualization of axonal transport.

PVH neurons of Long-Evans rat marked with retrograde tracer fluoro-gold

Techniques

Retrograde tracing can be achieved through various means, including the use of viral strains as markers of a cell’s connectivity to the injection site. The pseudorabies virus (PRV; Bartha strain), for example, may be used as a suitable tracer due to the propensity of the infection to spread upstream through a pathway of synaptically linked neurons, thus revealing the nature of their circuitry.[1][2]

Rabies has been shown to be effective for this system of circuit tracing because of its low level of damage to infected cells, specificity of infecting only neurons, and strict limitation of viral spread between neurons to synaptic regions.[3] These factors allow for highly specific traces that can reveal individual neuronal connections in a circuit without inflicting physical damage on the cells.

Another technique involves injecting special "beads" into the brain nuclei of anaesthetized animals.[4] The animals are allowed to survive for a few days and then euthanized. The cells in the origin of projection are visualized through an inverted fluorescence microscope.

A specialist technique was developed by Wickersham and colleagues, which employed a modified rabies virus. This virus was capable of infecting a single cell and jumping across one synapse; this allowed the researchers to investigate the local connectivity of neurons.[5]

Rabies virus

After being taken up at the synaptic terminal or axon of the target neuron, the rabies virus is enveloped in a vesicle which is transported towards the cell body via axonal dynein. In the wildtype rabies virus, the virus will continue to replicate and spread throughout the central nervous system until it has systemically infected the entire brain.[3] Deletion of the gene encoding glycoprotein (G protein) in rabies limits the spread of the virus strictly to cells that were initially infected. Transsynaptic spread of the virus can be limited to monosynaptic transmission to a neuron of origin by pseudotyping the G protein and putting the gene under Cre-control. This viral spread can be visualized through methods including addition of a fluorescence gene such as green fluorescent protein onto the viral cassette or through immunohistochemistry.[6][7]

Pseudorabies virus

A member of the herpesviridae family, the pseudorabies virus spreads through the CNS in both a retrograde and anterograde fashion, moving up the neural axon into the soma and dendrites in the retrograde application. Deletion of three key membrane protein genes in the PRV-Bartha strain of pseudorabies blocks anterograde spread of the virus and allows for additional manipulations to the viral DNA such as fluorescence to be added, allowing for retrograde circuit tracing.[8]

Fluoro-Gold

Fluoro-Gold, also known as hydroxystilbamidine, is a non-viral fluorescent retrograde tracer whose movement up the axon and across the dendritic tree can be visualized via fluorescent microscopy or immunohistochemistry.[9]

See also

References
  1. O'Donnell, P.; Lavín, A.; Enquist, L. W.; Grace, A. A.; Card, J. P. (1997). "Interconnected Parallel Circuits between Rat Nucleus Accumbens and Thalamus Revealed by Retrograde Transynaptic Transport of Pseudorabies Virus". Journal of Neuroscience. 17 (6): 2143–2167. doi:10.1523/jneurosci.17-06-02143.1997.
  2. Luo, A. H.; Aston-Jones, G. (2009). "Circuit projection from suprachiasmatic nucleus to ventral tegmental area: a novel circadian output pathway". European Journal of Neuroscience. 29 (4): 748–760. doi:10.1111/j.1460-9568.2008.06606.x. PMC 3649071. PMID 19200068.
  3. Davis, Benjamin M.; Rall, Glenn F.; Schnell, Matthias J. (2015-11-06). "Everything You Always Wanted to Know About Rabies Virus (But Were Afraid to Ask)". Annual Review of Virology. 2 (1): 451–71. doi:10.1146/annurev-virology-100114-055157. PMC 6842493. PMID 26958924.
  4. Katz, L. C.; Burkhalter, A.; Dreyer, W. J. (1984-08-09). "Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex". Nature. 310 (5977): 498–500. Bibcode:1984Natur.310..498K. doi:10.1038/310498a0. PMID 6205278.
  5. Wickersham IR, Lyon DC, Barnard RJ, et al. (March 2007). "Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons". Neuron. 53 (5): 639–47. doi:10.1016/j.neuron.2007.01.033. PMC 2629495. PMID 17329205.
  6. TotalBoox; TBX (2011-01-01). Research Advances in Rabies. Elsevier Science. ISBN 9780123870414. OCLC 968996286.
  7. Huang, Z. Josh; Zeng, Hongkui (2013-07-10). "Genetic Approaches to Neural Circuits in the Mouse". Annual Review of Neuroscience. 36: 183–215. doi:10.1146/annurev-neuro-062012-170307. PMID 23682658.
  8. Enquist, L. W. (2002-12-01). "Exploiting Circuit-Specific Spread of Pseudorabies Virus in the Central Nervous System: Insights to Pathogenesis and Circuit Tracers". The Journal of Infectious Diseases. 186 (Supplement_2): S209–S214. doi:10.1086/344278. ISSN 0022-1899. PMID 12424699.
  9. Naumann, T.; Härtig, W.; Frotscher, M. (2000-11-15). "Retrograde tracing with Fluoro-Gold: different methods of tracer detection at the ultrastructural level and neurodegenerative changes of back-filled neurons in long-term studies". Journal of Neuroscience Methods. 103 (1): 11–21. doi:10.1016/s0165-0270(00)00292-2. ISSN 0165-0270. PMID 11074092.

Further reading

Retrograde tracing has been extensively used in a broad array of neuroscience studies, including the following examples:

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