In last month’s column (“Painting Neural Circuitry With a Viral Brush,” Psychiatric Times, October 2008, page 16), I used Michelangelo’s famous fresco, “Hand of God Giving Life to Adam” on the ceiling of the Sistine Chapel as a metaphor to introduce a series of technologies that have allowed researchers to map the complex interactions of neural connections in continuously functioning neural tissues. This technology promises to deliver accurate synaptic associations—one finger to another—at a very high level of resolution
These extraordinary cartographic techniques involve exploiting the natural ability of the rabies virus to set up productive infections in neural tissues. For simplicity’s sake, we are examining the genetic manipulation of hypothetical “Neuron A” and its reaction to a previously engineered rabies virus. Although the manipulations to the virus are complex, using the data obtained, researchers seek to answer a simple, seemingly innocuous question: Are the neighbors green?
In case you do not have last month’s column handy, let me briefly review the life cycle of the rabies virus and reexamine the reengineered virus and Neuron A. We can then turn directly to the data.
As mentioned last month, the rabies virus has several biological aspects that make it an ideal delivery device for working with living neural tissues. Once inside a nerve cell, the virus sets up a manufacturing site to create more viruses, like any typical virus. At maturity, however, these progenies jump to neighboring neurons, which allows the virus to spread along specific neural routes. This life cycle is handy if you are interested in synaptic connections throughout the body. The infection can start in the peripheral nervous system and then jump the stout molecular border that separates it from the CNS. (That is why a bite anywhere on the body can result in a catastrophic brain infection.) If one could find a way to follow the virus, one could identify the routes by which it travels.
Aspects of this jumping ability were exploited in the circuit-mapping experiments we are about to review. Both virus and cell had to be genetically manipulated in 3 different ways for the experiment to work.
Glycoprotein gene deletion. First, a mutation was engineered that deleted a viral glycoprotein gene. This mutation rendered the virus incapable of spreading the infection beyond the first encountered cell, thereby stalling infection. If the virus were introduced to Neuron A, it could reproduce itself in Neuron A’s cytosol but would have no means of escaping Neuron A.
Artificial addition of EnvA gene. Second, a gene encoding the envelope protein EnvA from the avian sarcoma and leukosis virus (ASLV-A) was inserted into the manipulated viral genome. This allowed the virus, upon infection of Neuron A, to create the EnvA protein on its surface.
Why is this important? Putting the EnvA protein on the viral surface allows the virus to bind to and then enter any cell carrying EnvA’s natural receptor—a protein called TVA. Conveniently, human cells do not naturally possess TVA. Therefore, if you want this virus to infect a human cell, you are going to have to supply that cell with TVA artificially.
That change in function could be of great value to a neurobiologist. Suppose Neuron A, sporting its foreign EnvA, is embedded in a thicket of normal neurons that are not engineered in such fashion. If the hobbled virus were exposed to the entire thicket, the only cell that would become infected is Neuron A, not the neighbors.
With a one-two punch, a virus has been created that needs external help to set up a productive infection. It then needs further help if its progenies are going to infect the neighbors. The result? A “defanged” viral particle whose direction of infection can be manipulated. All that is needed is one more addition: an onboard tracking agent (such as a colorant) that would allow visual inspection of viral progress as the infection advanced.
Another artificial addition. Addition of just such a colorant was the third manipulation. The gene encoding a green fluorescent protein was also stitched into the rabies virus genome, which caused infected cells to glow green. Researchers could then detect the presence of infection simply by looking for the green protein.
Nerve cell modification. Next, Neuron A was engineered to interact with the rabies virus from the third manipulation. As mentioned last month, we are describing events in just Neuron A; however, the actual manipulation involved a large number of newborn rat cell hippocampal slices. In this experiment, the task was to engineer some of the neurons in such a fashion that the connec-tions between the modified neurons and their nonmanipulated neighbors could be discerned.