Viral neuronal tracing

A PC12 neuron infected with pseudorabies virus PRV GS443 (labeled in green). The green dots moving away from the cell body demonstrates anterograde transport.

Viral neuronal tracing is the use of a virus to trace neural pathways, providing a self-replicating tracer. Viruses have the advantage of self replication over molecular tracers, but can also spread too quickly and cause degradation of neural tissue. Viruses which can infect the nervous system, called neurotropic viruses, spread through spatially close assemblies of neurons through synapses, allowing for their use in studying functionally connected neural networks.[1][2]

The use of viruses to label functionally connected neurons stems from the work done by Albert Sabin, who developed a bioassay, which could assess the infection of viruses across neurons.[3] Subsequent research allowed for incorporation of immunohistochemical techniques to systematically label neuronal connections.[3] To date, viruses have been used to study multiple circuits in the nervous system.

History

Most neuroanatomists would agree that understanding how the brain is connected to itself and the body is of paramount importance.[4] As such, it is of equal importance to have a way to visualize and study the connections among neurons. Neuronal tracing methods offer an unprecedented view into the morphology and connectivity of neural networks. Depending on the tracer used, this can be limited to a single neuron or can progress trans-synaptically to adjacent neurons. After the tracer has spread sufficiently, the extent may be measured either by fluorescence (for dyes) or by immunohistochemistry (for biological tracers). An important innovation in this field is the use of neurotropic viruses as tracers. These not only spread throughout the initial site of infection, but can jump across synapses.

The use of a virus provides a self-replicating tracer. This allows for the elucidation of neural microcircuitry to an extent that was previously unobtainable. This has significant implications for the real world. A better understanding of the intimate connection between different parts of the brain leads to better prediction of the effect of localized brain injury. For example, if a patient has a stroke in the amygdala (primarily responsible for emotion), the patient might also have trouble learning to perform certain tasks because the amygdala is highly interconnected with the orbitofrontal cortex, which is responsible for reward learning.

In conclusion, to increase the hope of fixing brain injury, knowledge of its extent and complexity is key.[5]

Virus life cycle

The life cycle of viruses, such as those used in neuronal tracing, is different from cellular organisms. Viruses are parasitic in nature and cannot proliferate on their own. Therefore, they must infect another organism and effectively hijack cellular machinery to complete their life cycle.

The first stage of the viral life cycle is called viral entry. This defines the manner in which a virus infects a new host cell. In nature, neurotropic viruses are usually transmitted through bites or scratches, as in the case of rabies virus or certain strains of herpes viruses. In tracing studies, this step occurs artificially, typically through the use of a syringe. The next stage of the viral life cycle is called viral replication. During this stage, the virus takes over the host cell's machinery to cause the cell to create more viral proteins and assemble more viruses.

Once the cell has produced a sufficient number of viruses, the virus enters the viral shedding stage. During this stage, viruses leave the original host cell in search of a new host. In the case of neurotropic viruses, this transmission typically occurs at the synapse. Viruses can jump across the relatively short space from one neuron to the next. This trait is what makes viruses so useful in tracer studies.

Once the virus enters the next cell, the cycle begins anew. The original host cell begins to degrade after the shedding stage. In tracer studies, this is the reason the timing must be tightly controlled. If the virus is allowed to spread too far, the original microcircuitry of interest is degraded and no useful information can be retrieved. Typically, viruses can infect only a small number of organisms, and even then only a specific cell type within the body. The specificity of a particular virus for a specific tissue is known as its tropism. Viruses in tracer studies are all neurotropic (capable of infecting neurons).[6]

Methods

Infection

The viral tracer may be introduced in peripheral organs, such as a muscle or gland.[7] Certain viruses, such as adeno-associated virus, can be injected into the blood stream and can cross the blood–brain barrier to infect the brain.[8] It may also be introduced into a ganglion or injected directly into the brain using a stereotactic device. These methods offer unique insight into how the brain and its periphery are connected.

Viruses are introduced into neuronal tissue in many different ways. There are two major methods to introduce tracer into the target tissues.

  1. Pressure injection requires the tracer, in liquid form, to be injected directly into the cell. This is the most common method.
  2. Iontophoresis involves the application of current to the tracer solution within an electrode. The tracer molecules pick up a charge and are driven into the cell via the electric field. This is a useful method if you wish to label a cell after performing the patch clamp technique.[5]

Once the tracer is introduced into the cell, the aforementioned transport mechanisms take over. Then, the virus starts to infect cells in the local area onceit enters the nervous system. The viruses function by incorporating their own genetic material into the genome of the infected cells.[9] The host cell will then produce the proteins encoded by the gene. Researchers are able to incorporate numerous genes into the infected neurons, including fluorescent proteins used for visualization.[9] Further advances in neuronal tracing allow for targeted expression of fluorescent proteins to specific cell types.[9]

Histology and imaging

Once the virus has spread to the desired extent, the brain is sliced and mounted on slides. Then, fluorescent antibodies that are either specific for the virus, or fluorescent complementary DNA probes for viral DNA, are washed over the slices and imaged under a fluorescence microscope.[5]

Direction of transmission

Virus transmission relies on the mechanism of axoplasmic transport. Within the axon are long slender protein complexes called microtubules. They act as a cytoskeleton to help the cell maintain its shape. These can also act as highways within the axon and facilitate transport of neurotransmitter-filled vesicles and enzymes back and forth between the cell body, or soma and the axon terminal, or synapse.

Viruses can be transported in one of two directions: either anterograde (from soma to synapse), or retrograde (from synapse to soma). Neurons naturally transport proteins, neurotransmitters, and other macromolecules via these cellular pathways. Neuronal tracers, including viruses, take advantage of these transport mechanisms to distribute a tracer throughout a cell. Researchers can use this to study synaptic circuitry.

Anterograde transport

Anterograde tracing is the use of a tracer that moves from soma to synapse. Anterograde transport uses a protein called kinesin to move viruses along the axon in the anterograde direction.[5]

Retrograde transport

Retrograde tracing is the use of a tracer that moves from synapse to soma. Retrograde transport uses a protein called dynein to move viruses along the axon in the retrograde direction.[5][10] It is important to note that different tracers show characteristic affinities for dynein and kinesin, and so will spread at different rates.

Dual transport

At times, it is desirable to trace neurons upstream and downstream to determine both the inputs and the outputs of neural circuitry. This uses a combination of the above mechanisms.[11]

Benefits and drawbacks

Benefits

One of the benefits of using viral tracers is the ability of the virus to jump across synapses. This allows for tracing of microcircuitry as well as projection studies. Few molecular tracers are able to do this, and those that can usually have a decreased signal in secondary neurons, which leads to the other benefit of viral tracing - viruses can self-replicate. As soon as the secondary neuron is infected, the virus begins multiplying and replicating. There is no loss of signal as the tracer propagates through the brain.[6]

Drawbacks

As viruses propagate through the nervous system, the viral tracers infect neurons and ultimately destroy them. As such, the timing of tracer studies must be precise to allow adequate propagation before neural death occurs, causing large-scale harm to the body.

It has been difficult to find viruses perfectly suited for the task. A virus used for tracing should ideally be just mildly infectious to give good results, but not deadly as to destroy neural tissue too quickly or pose unnecessary risks to those exposed.

Another drawback is that viral neuronal tracing currently requires the additional step of attaching fluorescent antibodies to the viruses to visualise the path. In contrast, most molecular tracers are brightly colored and can be viewed with the naked eye, without additional modification.

Current uses

Viral tracing is primarily used to trace neuronal circuits. Researchers use one of the previously mentioned viruses to study how neurons in the brain are connected to each other with a very fine level of detail.[12] Connectivity largely determines how the brain functions. Viruses have been used to study retinal ganglion circuits,[13] cortical circuits,[14] and spinal circuits, among others.

Viruses in use

The following is a list of viruses currently in use for the purpose of neuronal tracing.

References

  1. ↑ Ugolini, Gabriella (2010). "Advances in viral transneuronal tracing". Journal of Neuroscience Methods. 194 (1): 2–20. doi:10.1016/j.jneumeth.2009.12.001. PMID 20004688. S2CID 43490041.
  2. ↑ Koyuncu, Orkide O.; Hogue, Ian B.; Enquist, Lynn W. (2013). "Virus Infections in the Nervous System". Cell Host & Microbe. 13 (4): 379–393. doi:10.1016/j.chom.2013.03.010. PMC 3647473. PMID 23601101.
  3. 1 2 Sams, J. M.; Jansen, A. S.; Mettenleiter, T. C.; Loewy, A. D. (1995-07-31). "Pseudorabies virus mutants as transneuronal markers". Brain Research. 687 (1–2): 182–190. doi:10.1016/0006-8993(95)00484-8. ISSN 0006-8993. PMID 7583303. S2CID 21516719.
  4. ↑ Perkel, Jeffrey M. (2013-01-18). "LIFE SCIENCE TECHNOLOGIES: This Is Your Brain: Mapping the Connectome". Science. 339 (6117): 350–352. Bibcode:2013Sci...339..350P. doi:10.1126/science.339.6117.350. ISSN 0036-8075.
  5. 1 2 3 4 5 Oztas E (2003). "Neuronal Tracing". Neuroanatomy. 2: 2–5.
  6. 1 2 Ginger, Melanie; Bony, Guillaume; Haberl, Matthias; Frick, Andreas (2014-10-28). Biology and Pathogenesis of Rhabdo- and Filoviruses. WORLD SCIENTIFIC. pp. 263–287. doi:10.1142/9789814635349_0011. ISBN 9789814635332.
  7. ↑ Ugolini G (1995). "Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups. [Research Support, Non-U.S. Gov't]". J Comp Neurol. 356 (3): 457–480. doi:10.1002/cne.903560312. PMID 7642806. S2CID 519638.
  8. ↑ "Injection sends 'genetic cargo' to neurons all over the body - Futurity". Futurity. 2017-06-29. Retrieved 2018-04-01.
  9. 1 2 3 Callaway, Edward M (2008). "Transneuronal circuit tracing with neurotropic viruses". Current Opinion in Neurobiology. 18 (6): 617–623. doi:10.1016/j.conb.2009.03.007. PMC 2698966. PMID 19349161.
  10. ↑ Wickersham I. R.; Finke S.; Conzelmann K. K.; Callaway E. M. (2007). "Retrograde neuronal tracing with a deletion-mutant rabies virus. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]". Nat Methods. 4 (1): 47–49. doi:10.1038/nmeth999. PMC 2755236. PMID 17179932.
  11. ↑ Lopez I. P.; Salin P.; Kachidian P.; Barroso-Chinea P.; Rico A. J.; Gomez-Bautista V.; Lanciego J. L. (2010). "The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing. [Research Support, Non-U.S. Gov't]". J Neurosci Methods. 194 (1): 21–27. doi:10.1016/j.jneumeth.2010.01.015. PMID 20096304. S2CID 214343.
  12. 1 2 Ginger M.; Haberl M.; Conzelmann K.-K.; Schwarz M.; Frick A. (2013). "Revealing the secrets of neuronal circuits with recombinant rabies virus technology. [Research Support, Non-U.S. Gov't Review]". Front. Neural Circuits. 7: 2. doi:10.3389/fncir.2013.00002. PMC 3553424. PMID 23355811.
  13. ↑ Viney T. J.; Balint K.; Hillier D.; Siegert S.; Boldogkoi Z.; Enquist L. W.; Roska B. (2007). "Local retinal circuits of melanopsin-containing ganglion cells identified by transsynaptic viral tracing. [Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.]". Curr Biol. 17 (11): 981–988. doi:10.1016/j.cub.2007.04.058. PMID 17524644. S2CID 2388142.
  14. ↑ Ugolini G (2011). "Rabies virus as a transneuronal tracer of neuronal connections. [Research Support, Non-U.S. Gov't Review]". Adv Virus Res. 79: 165–202. doi:10.1016/B978-0-12-387040-7.00010-X. PMID 21601048.
  15. ↑ McGovern AE, Davis-Poynter N, Rakoczy J, Phipps S, Simmons DG, Mazzone SB.; Davis-Poynter; Rakoczy; Phipps; Simmons; Mazzone (Jul 30, 2012). "Anterograde neuronal circuit tracing using a genetically modified herpes simplex virus expressing EGFP". J Neurosci Methods. 209 (1): 158–67. doi:10.1016/j.jneumeth.2012.05.035. PMID 22687938. S2CID 20370171.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  16. ↑ Norgren, R. B., Jr., & Lehman, M. N.; Lehman (1998). "Herpes simplex virus as a transneuronal tracer. [Review]". Neurosci Biobehav Rev. 22 (6): 695–708. doi:10.1016/s0149-7634(98)00008-6. PMID 9809305. S2CID 40884240.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ↑ Koyuncu OO, Perlman DH, Enquist LW; Perlman; Enquist (Jan 16, 2013). "Efficient retrograde transport of pseudorabies virus within neurons requires local protein synthesis in axons". Cell Host Microbe. 13 (1): 54–66. doi:10.1016/j.chom.2012.10.021. PMC 3552305. PMID 23332155.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  18. ↑ Kratchmarov R, Taylor MP, Enquist LW; Taylor; Enquist (2013). "Role of us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus". PLOS ONE. 8 (3): e58776. Bibcode:2013PLoSO...858776K. doi:10.1371/journal.pone.0058776. PMC 3602541. PMID 23527020.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  19. ↑ Kelly, R. M., & Strick, P. L.; Strick (2000). "Rabies as a transneuronal tracer of circuits in the central nervous system. [Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. Review]". J Neurosci Methods. 103 (1): 63–71. doi:10.1016/S0165-0270(00)00296-X. PMID 11074096. S2CID 17492937.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  20. ↑ Ugolini, G. (2008). "Use of rabies virus as a transneuronal tracer of neuronal connections: implications for the understanding of rabies pathogenesis. [Research Support, Non-U.S. Gov't Review]". Dev Biol (Basel). 131: 493–506. PMID 18634512.
  21. ↑ Beier K. T.; Saunders A.; Oldenburg I. A.; Miyamichi K.; Akhtar N.; Luo L.; Whelang SPJ; Sabatini B; Cepko C. L. (2011). "Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors". Proc Natl Acad Sci U S A. 108 (37): 15414–15419. doi:10.1073/pnas.1110854108. PMC 3174680. PMID 21825165.
  22. ↑ Beier KT, Saunders AB, Oldenburg IA, Sabatini BL, Cepko CL; Saunders; Oldenburg; Sabatini; Cepko (2013). "Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo". Frontiers in Neural Circuits. 7 (11): 1–13. doi:10.3389/fncir.2013.00011. PMC 3566411. PMID 23403489.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  23. ↑ Beier KT, Saunders AB, Oldenburg IA, Sabatini BL, Cepko CL; Tomioka; Taki; Nakamura; Tamamaki; Kaneko (2001). "In Vivo Transduction of Central Neurons Using Recombinant Sindbis Virus: Golgi-like Labeling of Dendrites and Axons with Membrane-targeted Fluorescent Proteins". The Journal of Histochemistry and Cytochemistry. 49 (12): 1497–1507. doi:10.1177/002215540104901203. PMID 11724897.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  24. ↑ Chamberlin NL, Du B, de Lacalle S, Saper CB; Du; De Lacalle; Saper (May 18, 1998). "Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS". Brain Res. 793 (1–2): 169–75. doi:10.1016/s0006-8993(98)00169-3. PMC 4961038. PMID 9630611.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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