Retrograde tracing

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

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.

Contents

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

Related Research Articles

The development of the nervous system, or neural development, or neurodevelopment, refers to the processes that generate, shape, and reshape the nervous system of animals, from the earliest stages of embryonic development to adulthood. The field of neural development draws on both neuroscience and developmental biology to describe and provide insight into the cellular and molecular mechanisms by which complex nervous systems develop, from nematodes and fruit flies to mammals.

Neuroanatomy Branch of neuroscience

Neuroanatomy is the study of the structure and organization of the nervous system. In contrast to animals with radial symmetry, whose nervous system consists of a distributed network of cells, animals with bilateral symmetry have segregated, defined nervous systems. Their neuroanatomy is therefore better understood. In vertebrates, the nervous system is segregated into the internal structure of the brain and spinal cord and the routes of the nerves that connect to the rest of the body. The delineation of distinct structures and regions of the nervous system has been critical in investigating how it works. For example, much of what neuroscientists have learned comes from observing how damage or "lesions" to specific brain areas affects behavior or other neural functions.

A histochemical tracer is a compound used to reveal the location of cells and track neuronal projections. A neuronal tracer may be retrograde, anterograde, or work in both directions. A retrograde tracer is taken up in the terminal of the neuron and transported to the cell body, whereas an anterograde tracer moves away from the cell body of the neuron.

Behavioral neuroscience, also known as biological psychology, biopsychology, or psychobiology, is the application of the principles of biology to the study of physiological, genetic, and developmental mechanisms of behavior in humans and other animals.

Rabies virus Species of virus

Rabies virus, scientific name Rabies lyssavirus, is a neurotropic virus that causes rabies in humans and animals. Rabies transmission can occur through the saliva of animals and less commonly through contact with human saliva. Rabies lyssavirus, like many rhabdoviruses, has an extremely wide host range. In the wild it has been found infecting many mammalian species, while in the laboratory it has been found that birds can be infected, as well as cell cultures from mammals, birds, reptiles and insects. Rabies is reported in more than 150 countries on all continents, with the exclusion of Antarctica. The main burden of disease is reported in Asia and Africa, but some cases have been reported also in Europe in the past 10 years, especially in returning travellers.

Neural circuit Network or circuit of neurons

A neural circuit is a population of neurons interconnected by synapses to carry out a specific function when activated. Neural circuits interconnect to one another to form large scale brain networks. Biological neural networks have inspired the design of artificial neural networks, but artificial neural networks are usually not strict copies of their biological counterparts.

Hydroxystilbamidine Chemical compound

Hydroxystilbamidine is a fluorescent dye that emits different frequencies of light when bound to DNA and RNA. It is used as a retrograde tracer for outlining neurons, and as a histochemical stain.

Axonal transport

Axonal transport, also called axoplasmic transport or axoplasmic flow, is a cellular process responsible for movement of mitochondria, lipids, synaptic vesicles, proteins, and other organelles to and from a neuron's cell body, through the cytoplasm of its axon called the axoplasm. Since some axons are on the order of meters long, neurons cannot rely on diffusion to carry products of the nucleus and organelles to the end of their axons. Axonal transport is also responsible for moving molecules destined for degradation from the axon back to the cell body, where they are broken down by lysosomes.

Aujeszky's disease, usually called pseudorabies in the United States, is a viral disease in swine that has been endemic in most parts of the world. It is caused by Suid herpesvirus 1 (SuHV-1). Aujeszky's disease is considered to be the most economically important viral disease of swine in areas where classical swine fever has been eradicated. Other mammals, such as cattle, sheep, goats, cats, dogs, and raccoons, are also susceptible. The disease is usually fatal in these animal species.

Brainbow Neuroimaging technique to differentiate neurons

Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of neural connectomics.

Optogenetics is a biological technique that involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels. As such, optogenetics is a neuromodulation method that uses a combination of techniques from optics and genetics to control the activities of individual neurons in living tissue — even within freely-moving animals. In some usages, optogenetics also refers to optical monitoring of neuronal activity or control of other biochemical pathways in non-neuronal cells, although these research activities preceded the use of light-sensitive ion channels in neurons. As optogenetics is used by some authors to refer to only optical control of the activity of genetically defined neurons and not these additional research approaches, the term optogenetics is an example of polysemy.

Rabies Deadly viral disease, transmitted through animals

Rabies is a viral disease that causes inflammation of the brain in humans and other mammals. Early symptoms can include fever and tingling at the site of exposure. These symptoms are followed by one or more of the following symptoms: nausea, vomiting, violent movements, uncontrolled excitement, fear of water, an inability to move parts of the body, confusion, and loss of consciousness. Once symptoms appear, the result is nearly always death. The time period between contracting the disease and the start of symptoms is usually one to three months but can vary from less than one week to more than one year. The time depends on the distance the virus must travel along peripheral nerves to reach the central nervous system.

In neuroscience, anterograde tracing is a research method which is used to trace axonal projections from their source to their point of termination. The complementary technique is retrograde tracing, which is used to trace neural connections from their termination to their source. Both the anterograde and retrograde tracing techniques are based on the visualization of the biological process of axonal transport.

Transneuronal degeneration is the death of neurons resulting from the disruption of input from or output to other nearby neurons. It is an active excitotoxic process when a neuron is overstimulated by a neurotransmitter causing the dysfunction of that neuron which drives neighboring neurons into metabolic deficit, resulting in rapid, widespread loss of neurons. This can be either anterograde or retrograde, indicating the direction of the degeneration relative to the original site of damage. There are varying causes for transneuronal degeneration such as brain lesions, disconnection syndromes, respiratory chain deficient neuron interaction, and lobectomies. Although there are different causes, transneuronal degeneration generally results in the same effects to varying degrees. Transneuronal degeneration is thought to be linked to a number of diseases, most notably Huntington's disease and Alzheimer's disease, and researchers recently have been performing experiments with monkeys and rats, monitoring lesions in different parts of the body to study more closely how exactly the process works.

Developmental plasticity is a general term referring to changes in neural connections during development as a result of environmental interactions as well as neural changes induced by learning. Much like neuroplasticity or brain plasticity, developmental plasticity is specific to the change in neurons and synaptic connections as a consequence of developmental processes. A child creates most of these connections from birth to early childhood.

Neurovirology is an interdisciplinary field which represents a melding of clinical neuroscience, virology, immunology, and molecular biology. The main focus of the field is to study viruses capable of infecting the nervous system. In addition to this, the field studies the use of viruses to trace neuroanatomical pathways, for gene therapy, and to eliminate detrimental populations of neural cells.

In neuroscience, synaptic scaling is a form of homeostatic plasticity, in which the brain responds to chronically elevated activity in a neural circuit with negative feedback, allowing individual neurons to reduce their overall action potential firing rate. Where Hebbian plasticity mechanisms modify neural synaptic connections selectively, synaptic scaling normalizes all neural synaptic connections by decreasing the strength of each synapse by the same factor, so that the relative synaptic weighting of each synapse is preserved.

Viral neuronal tracing

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

Neuronal tracing, or neuron reconstruction is a technique used in neuroscience to determine the pathway of the neurites or neuronal processes, the axons and dendrites, of a neuron. From a sample preparation point of view, it may refer to some of the following as well as other genetic neuron labeling techniques,

An autapse is a chemical or electrical synapse from a neuron onto itself. It can also be described as a synapse formed by the axon of a neuron on its own dendrites, in vivo or in vitro.

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. 1 2 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. S2CID   36191957.
  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. S2CID   24155326.

Further reading

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