Anterograde tracing
Encyclopedia
In neuroscience
, anterograde tracing is a research method which is used to trace axonal projections from their source (the ell body] or soma]) to their point of termination (the synapse
). The complementary technique is retrograde tracing, which is used to trace neural connections from their termination to their source (i.e. synapse to cell body). Both the anterograde and retrograde tracing techniques are based on the visualization of the biological process
of axonal transport.
The anterograde and retrograde tracing techniques allow the detailed descriptions of neuronal projections from a single population of neurons to their various targets 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. Much of what is currently known about connectional neuroanatomy
was discovered through the use of the anterograde and retrograde tracing techniques.
s (e.g. Green fluorescent protein
, lipophylic dyes or radioactively tagged amino acids) into the brain
. These molecules are absorbed locally by the soma
(cell body) of various neurons and transported to the axon terminal
s, or they are absorbed by axon
s and transported to the soma of the neuron. Other tracer molecules allow for the visualization of large networks of axonal projections extending from the neurons exposed to the tracer.
The aforementioned tracers such as GFP and DiI are technically not anterograde tracers: they do not selectively travel in a certain direction and are not actively transported by the cell. Furthermore, they do not cross the synaptic cleft, and therefore only label the neurons that were directly at the site of tracer application. These dyes are mostly used as a filler to mark cells that have been recorded using electrophysiology
: this allows the cells that have been recorded to also be morphologically reconstructed in 3D.
The anterograde tracers that can cross the synaptic cleft and label multiple neurons within a pathway can be divided in two categories: genetic and molecular tracers.
type1 (HSV) and the Rhabdoviruses
. HSV was used to trace the connections between the brain and the stomach, in order to examine the brain areas involved in viscero-sensory processing. Another study used HSV type1 and type2 to investigate the optical pathway: by injecting the virus into the eye, the pathway from the retina
into the brain was visualized. A specialist technique was developed by Wickersham and colleagues, which employed a modified Rabies
virus. This virus was only capable of infecting a single cell, and only capable of jumping across 1 synapse; this allowed the researchers to investigate the local connectivity of neurons.
Viral tracers use a receptor on the host cell to attach to it and are then endocytosed
. For example, HSV uses the nectin
receptor and is then endocytosed. After endocytosis, the low pH inside the vesicle strips the envelope of the virion after which the virus is ready to be transported to the cell body. It was shown that pH and endocytosis are crucial for the HSV to infect a cell. Transport of the viral particles along the axon was shown to depend on the microtubular cytoskeleton
.
s,and is then taken up via endocytosis via a caveolae-dependent pathway.
Other anterograde tracers widely used in neuroanatomy are the biotinylated dextran amine
s (BDA), also used in retrograde labeling
.
Neuroscience
Neuroscience is the scientific study of the nervous system. Traditionally, neuroscience has been seen as a branch of biology. However, it is currently an interdisciplinary science that collaborates with other fields such as chemistry, computer science, engineering, linguistics, mathematics,...
, anterograde tracing is a research method which is used to trace axonal projections from their source (the ell body] or soma]) to their point of termination (the synapse
Synapse
In the nervous system, a synapse is a structure that permits a neuron to pass an electrical or chemical signal to another cell...
). The complementary technique is retrograde tracing, which is used to trace neural connections from their termination to their source (i.e. synapse to cell body). Both the anterograde and retrograde tracing techniques are based on the visualization of the biological process
Biological process
A biological process is a process of a living organism. Biological processes are made up of any number of chemical reactions or other events that results in a transformation....
of axonal transport.
The anterograde and retrograde tracing techniques allow the detailed descriptions of neuronal projections from a single population of neurons to their various targets throughout the nervous system
Nervous system
The nervous system is an organ system containing a network of specialized cells called neurons that coordinate the actions of an animal and transmit signals between different parts of its body. In most animals the nervous system consists of two parts, central and peripheral. The central nervous...
. These techniques allow the "mapping" of connections between neurons in a particular structure (e.g. the eye
Human eye
The human eye is an organ which reacts to light for several purposes. As a conscious sense organ, the eye allows vision. Rod and cone cells in the retina allow conscious light perception and vision including color differentiation and the perception of depth...
) and the target neurons in the brain. Much of what is currently known about connectional neuroanatomy
Neuroanatomy
Neuroanatomy is the study of the anatomy 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, and thus we can begin to speak of...
was discovered through the use of the anterograde and retrograde tracing techniques.
Techniques
Several methods exist to trace projections originating from the soma towards their target areas. These techniques initially relied upon the direct physical injection of various visualizable tracer moleculeMolecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
s (e.g. Green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
, lipophylic dyes or radioactively tagged amino acids) into the brain
Brain
The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...
. These molecules are absorbed locally by the soma
Soma (biology)
The soma , or perikaryon , or cyton, is the bulbous end of a neuron, containing the cell nucleus. The word "soma" comes from the Greek σῶμα, meaning "body"; the soma of a neuron is often called the "cell body"...
(cell body) of various neurons and transported to the axon terminal
Axon terminal
Axon terminals are distal terminations of the branches of an axon. An axon nerve fiber is a long, slender projection of a nerve cell, or neuron, that conducts electrical impulses away from the neuron's cell body, or soma, in order to transmit those impulses to other neurons.Neurons are...
s, or they are absorbed by axon
Axon
An axon is a long, slender projection of a nerve cell, or neuron, that conducts electrical impulses away from the neuron's cell body or soma....
s and transported to the soma of the neuron. Other tracer molecules allow for the visualization of large networks of axonal projections extending from the neurons exposed to the tracer.
The aforementioned tracers such as GFP and DiI are technically not anterograde tracers: they do not selectively travel in a certain direction and are not actively transported by the cell. Furthermore, they do not cross the synaptic cleft, and therefore only label the neurons that were directly at the site of tracer application. These dyes are mostly used as a filler to mark cells that have been recorded using electrophysiology
Electrophysiology
Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage change or electric current on a wide variety of scales from single ion channel proteins to whole organs like the heart...
: this allows the cells that have been recorded to also be morphologically reconstructed in 3D.
The anterograde tracers that can cross the synaptic cleft and label multiple neurons within a pathway can be divided in two categories: genetic and molecular tracers.
Genetic tracers
In order to trace projections from a specific region or cell, a genetic construct, virus or protein can be locally injected, after which it is allowed to be transported anterogradely. Viral tracers can cross the synapse, and can be used to trace connectivity between brain regions across many synapses. Examples of viruses used for anterograde tracing are described by Kuypers. Most well known are the Herpes simplex virusHerpes simplex virus
Herpes simplex virus 1 and 2 , also known as Human herpes virus 1 and 2 , are two members of the herpes virus family, Herpesviridae, that infect humans. Both HSV-1 and HSV-2 are ubiquitous and contagious...
type1 (HSV) and the Rhabdoviruses
Rhabdoviridae
Rhabdoviruses are viruses belonging to the family Rhabdoviridae, which is in the order Mononegavirales. The name is derived from the Greek rhabdos meaning rod referring to the shape of the viral particles. Rhabdoviruses infect a broad range of hosts throughout the animal and plant kingdoms...
. HSV was used to trace the connections between the brain and the stomach, in order to examine the brain areas involved in viscero-sensory processing. Another study used HSV type1 and type2 to investigate the optical pathway: by injecting the virus into the eye, the pathway from the retina
Retina
The vertebrate retina is a light-sensitive tissue lining the inner surface of the eye. The optics of the eye create an image of the visual world on the retina, which serves much the same function as the film in a camera. Light striking the retina initiates a cascade of chemical and electrical...
into the brain was visualized. A specialist technique was developed by Wickersham and colleagues, which employed a modified Rabies
Rabies
Rabies is a viral disease that causes acute encephalitis in warm-blooded animals. It is zoonotic , most commonly by a bite from an infected animal. For a human, rabies is almost invariably fatal if post-exposure prophylaxis is not administered prior to the onset of severe symptoms...
virus. This virus was only capable of infecting a single cell, and only capable of jumping across 1 synapse; this allowed the researchers to investigate the local connectivity of neurons.
Viral tracers use a receptor on the host cell to attach to it and are then endocytosed
Endocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
. For example, HSV uses the nectin
Nectin
Nectins and Nectin-like molecules are families of cellular adhesion molecules involved in Ca2+-independent cellular adhesion.Nectins are ubiquitously expressed and have adhesive roles in a wide range of tissues such as the adherens junction of epithelia or the chemical synapse of the neuronal...
receptor and is then endocytosed. After endocytosis, the low pH inside the vesicle strips the envelope of the virion after which the virus is ready to be transported to the cell body. It was shown that pH and endocytosis are crucial for the HSV to infect a cell. Transport of the viral particles along the axon was shown to depend on the microtubular cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
.
Molecular tracers
There is also a group of tracers that consist of protein products that can be taken up by the cell and transported across the synapse into the next cell. Wheat-germ agglutinin (WGA) and Phaseolus vulgaris leucoagglutinin are the most well known tracers, however they are not strict anterograde tracers: especially WGA is known to be transported anterogradely as well as retrogradely. WGA enters the cell by binding to OligosaccharideOligosaccharide
An oligosaccharide is a saccharide polymer containing a small number of component sugars, also known as simple sugars...
s,and is then taken up via endocytosis via a caveolae-dependent pathway.
Other anterograde tracers widely used in neuroanatomy are the biotinylated dextran amine
Biotinylated dextran amine
Biotinylated dextran amines are organic compounds used as anterograde and retrograde neuroanatomical tracers. They can be used for labeling the source as well as the point of termination of neural connections and therefore to study neural pathways....
s (BDA), also used in retrograde labeling
Retrograde tracing
Retrograde tracing is a research method which is used in neuroscience to trace neural connections from their point of termination to their source . The opposite technique is anterograde tracing, which is used to trace neural connections from their source to their point of termination...
.