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 disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, development ...
to trace neural connections from their point of termination (the
synapse
In the nervous system, a synapse is a structure that permits a neuron (or nerve cell) to pass an electrical or chemical signal to another neuron or to the target effector cell.
Synapses are essential to the transmission of nervous impulses from ...
) 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 highly complex part of an animal that coordinates its actions and sensory information by transmitting signals to and from different parts of its body. The nervous system detects environmental changes th ...
. These techniques allow the "mapping" of connections between neurons in a particular structure (e.g. the
eye) and the target neurons in the
brain
A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a v ...
. 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
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 cytopla ...
.
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
brain
A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a v ...
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. "Fluorescence microsc ...
.
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. 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, vi ...
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 or
axon
An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences), is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action po ...
of the target neuron, the
rabies 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 lys ...
is enveloped in a
vesicle
Vesicle may refer to:
; In cellular biology or chemistry
* Vesicle (biology and chemistry)
In cell biology, a vesicle is a structure within or outside a cell, consisting of liquid or cytoplasm enclosed by a lipid bilayer. Vesicles form nat ...
which is transported towards the cell body via axonal
dynein. 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 onto the viral
cassette
Cassette may refer to:
Technology
* Cassette tape (or ''musicassette'', ''audio cassette'', ''cassette tape'', or ''tape''), a worldwide standard for analog audio recording and playback
** Cassette single (or "Cassingle"), a music single in th ...
or through
immunohistochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to an ...
.
Pseudorabies virus
A member of the
herpesviridae
''Herpesviridae'' is a large family of DNA viruses that cause infections and certain diseases in animals, including humans. The members of this family are also known as herpesviruses. The family name is derived from the Greek word ''ἕρπει ...
family, the
pseudorabies
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 mos ...
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
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
Histology,
also known as microscopic anatomy o ...
, 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.
See also
*
Anterograde tracing
*
Neural pathway
In neuroanatomy, a neural pathway is the connection formed by axons that project from neurons to make synapses onto neurons in another location, to enable neurotransmission (the sending of a signal from one region of the nervous system to an ...
*
Viral neuronal tracing
References
Further reading
Retrograde tracing has been extensively used in a broad array of neuroscience studies, including the following examples:
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{{DEFAULTSORT:Retrograde Tracing
Neural coding