Retrograde tracing is a research method used in

neuroscience Neuroscience is the scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions, and its disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, ...

to trace neural connections from their point of termination (the

synapse In the nervous system, a synapse is a structure that allows a neuron (or nerve cell) to pass an electrical or chemical signal to another neuron or a target effector cell. Synapses can be classified as either chemical or electrical, depending o ...

) 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 In biology, the nervous system is the complex system, highly complex part of an animal that coordinates its behavior, actions and sense, sensory information by transmitting action potential, signals to and from different parts of its body. Th ...

. These techniques allow the "mapping" of connections between neurons in a particular structure (e.g. the

eye An eye is a sensory organ that allows an organism to perceive visual information. It detects light and converts it into electro-chemical impulses in neurons (neurones). It is part of an organism's visual system. In higher organisms, the ey ...

) and the target neurons in the

brain The brain is an organ (biology), organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It consists of nervous tissue and is typically located in the head (cephalization), usually near organs for ...

. The opposite technique is

anterograde tracing 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 p ...

, 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.

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. 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. 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

nuclei of anaesthetized animals. 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 fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. A fluorescence micro ...

. A specialist technique was developed by Wickersham and colleagues, which employed a modified

rabies Rabies is a viral disease that causes encephalitis in humans and other mammals. It was historically referred to as hydrophobia ("fear of water") because its victims panic when offered liquids to drink. Early symptoms can include fever and abn ...

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.

Rabies virus

After being taken up at the

synaptic terminal Chemical synapses are biological junctions through which neurons' signals can be sent to each other and to non-neuronal cells such as those in muscles or glands. Chemical synapses allow neurons to form circuits within the central nervous syste ...

axon An axon (from Greek ἄξων ''áxōn'', axis) or nerve fiber (or nerve fibre: see American and British English spelling differences#-re, -er, spelling differences) is a long, slender cellular extensions, projection of a nerve cell, or neuron, ...

of the target neuron, the

rabies virus Rabies virus (''Lyssavirus rabies'') is a neurotropic virus that causes rabies in animals, including humans. It can cause violence, hydrophobia, and fever. Rabies transmission can also occur through the saliva of animals and less commonly throu ...

is enveloped in a vesicle which is transported towards the cell body via axonal

dynein Dyneins are a family of cytoskeletal motor proteins (though they are actually protein complexes) that move along microtubules in cells. They convert the chemical energy stored in ATP to mechanical work. Dynein transports various cellular cargo ...

. In the

wildtype The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

rabies virus, the virus will continue to replicate and spread throughout the central nervous system until it has systemically infected the entire brain. 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 The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victo ...

onto the viral cassette or through

immunohistochemistry Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of Antibody, antibodies binding specifically to antigens in biological tissues. Alber ...

Pseudorabies virus

A member of the

herpesviridae ''Orthoherpesviridae'', previously named and more widely known as ''Herpesviridae'', is a large family of DNA viruses that cause infections and certain diseases in animals, including humans. The members of this family are commonly known as herp ...

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.

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.

Techniques

Rabies virus

Pseudorabies virus

Fluoro-Gold

See also

References

Further reading