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Viral neuronal tracing is the use of a virus to trace neural pathways, providing a self-replicating tracer.

Background of Neural Tracing
Most neuroanatomists would agree that understanding how the brain is connected to itself and the body is of paramount importance. 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 can allow for the elucidation of neural microcircuitry to an extent that was previously unobtainable. This has significant implications for the real world. If we can better understand what parts of the brain are intimately connected, we can predict 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, responsible for reward learning. As always, the first step to solving a problem is fully understanding it, so if we are to have any hope of fixing brain injury, we must first understand its extent and complexity.

Direction of transmission
Viruses can be transmitted in one of two directions. First, one must understand the underlying 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. Transport can proceed in either direction: 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.

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,. 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.

Infection
The viral tracer may be introduced in peripheral organs, such as a muscle or gland. 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. Pressure injection requires the tracer, in liquid form, to be injected directly into the cell. This is the most common method. 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. Once the tracer is introduced into the cell, the aforementioned transport mechanisms take over.

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

Benefits and Drawbacks
The use of viruses as tracers has its benefits and its drawbacks. As such, there are some applications in which viruses are an excellent tracer, and other applications in which there are better methods to use.

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. Therefore, another benefit of viral tracing is the ability of viruses to 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.

Drawbacks
One of the drawbacks of viruses is, ironically, the fact that they are viruses. As they propagate through the nervous system, the viral tracers infect neurons and ultimately destroy them. Therefore, the timing of tracer studies must be precise to allow adequate propagation before neural death occurs. The viruses are not only harmful to neural tissue, many are also harmful to humans. Therefore, it has been a difficult task to find adequate viruses for the task. Any virus used for tracing must be just infectious enough to give good results, but must not destroy neural tissue too quickly or pose unnecessary risk to the researchers involved. Yet another drawback is that they must be visualized with fluorescent antibodies, adding an additional step to processing. In contrast, most molecular tracers are brightly colored and can be viewed with the naked eye.

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. Connectivity largely determines how the brain functions. Viruses have been used to study retinal ganglion circuits, cortical circuits , and spinal circuits, among others.

Viruses in use
The following is a list of viruses currently in use for the purpose of neuronal tracing. The most common viruses in use are the pseudorabies virus and the herpes simplex virus.
 * Pseudorabies virus
 * Herpes simplex virus
 * Vesicular stomatitis virus