Virus leaves trail in brain for researchers
Princeton NJ -- Research by Princeton biologists has shown how a virus usually known for its potent attacks on the brain can be turned into a tool for mapping and characterizing its former battleground.
Lynn Enquist, a professor of molecular biology, and colleagues in his lab and at Rockefeller University developed a genetically engineered virus that infects only nerve cells that are joined to each other in the same neural circuit. The virus carries with it a fluorescent marker that essentially draws a map of the brain's wiring. The achievement gives neuroscientists an unprecedented tool for understanding how the billions of neurons in the brain work together to perform complex tasks.
"These are unbelievably small, self-amplifying circuit tracers," said Enquist. "It's like putting a dropperful of something on the tail light of your car and watching it light up the battery and all the wires in between."
In a paper published in the March 30 issue of Science, Enquist and graduate student Mark Tomishima collaborated with scientists at Rockefeller University in New York and the University of California-San Diego to demonstrate the system by mapping neural circuits related to appetite control in mice.
The researchers injected the genetically engineered virus into mouse brain regions that are known to handle aspects of appetite control. Tracing the progress of infection, they found connections between these appetite centers and several other parts of the brain, including those devoted to the highest levels of reasoning.
Although this particular result does not by itself give major insights into appetite control, it is a clear demonstration of how scientists can investigate this and other brain functions. Further study of the brain areas connected by these newly traced neurons could lead, for example, to the discovery of brain chemicals that control feeding. Such chemicals would be of great interest to pharmaceutical companies developing weight-loss drugs.
"These viruses are really telling us something about how the brain is wired," said Enquist.
"It is an enormous advance for those of us who are concerned about the circuitry of the brain," commented Clifford Saper, a Harvard University professor of neuroscience and chair of neurology at Beth Israel Deaconesss Medical Center in Boston. "We are already gearing up to import it into our own lab to solve a variety of problems that have bugged us for years."
What makes the technique so powerful is that the virus has been engineered to infect only a particular class of cells. Earlier work by Enquist and others have shown the potential for a herpes virus called pseudorabies to trace nerve circuits, but the researchers were not able to exercise much control over where the tracing began. Enquist had used the virus, for example, to map circuits from both the stomach and the eye to the brain.
The limitation was that when the virus was injected, it infected nerve cells indiscriminately, so that it traced whatever circuits happened to be close to the injection site. That fact limited the usefulness of injecting the virus into the brain because even a very small injection into a very limited area would result in the infection of many circuits, with no practical way of discriminating one traced circuit from another.
In response to a suggestion and encouragement from molecular geneticist Jeffrey Friedman at Rockefeller, Enquist and Tomishima worked with Friedman's postdoctoral fellow Jeff DeFalco to develop a more sophisticated version of the pseudorabies virus. Their new system requires two parts. First they deleted a key gene that the virus requires for replicating itself. They replaced it with a new version that works only in cells containing a counterpart gene called cre. The second step, developed by Friedman and his colleagues, was to engineer a strain of mouse that carries the cre gene. The trick was to rig the cre gene so that it only works in brain cells that produce proteins known to be involved in appetite control.
The result is that when the engineered virus enters the engineered mouse, it only replicates in cells that produce the appetite proteins. More important, the replicating virus now spreads to only those neurons that are connected to the originally infected neuron.
For their first study using the new tool, the researchers focused on two appetite-related proteins called leptin and neuropeptide Y, which have been the subject of intense commercial and academic interest for their potential use in developing weight-loss drugs.
For Enquist, the development of viral tracers is a sidelight to his main research into understanding how viruses infect and damage the brain, and how the brain responds to such invasions. His work has focused on such viruses as pseudorabies, which causes deadly infections in animals, herpes simplex, which produces a range of illnesses in humans, and varicella-zoster, which causes chickenpox and shingles.
Although it is a departure from his primary work, the viral tracer research may ultimately come full circle and offer clues to the progression and possible treatment of brain infections. "If we know how the virus travels from one area to another, we can look and see how these areas are mounting a defense," said Enquist.
Besides, he said, "This cottage industry of building viral tracers is getting to be a lot of fun too."
As a next step, Enquist and colleagues hope to incorporate more control mechanisms into the virus. Recent advances in biotechnology have produced several tools for controlling gene activity beyond the cre gene used in the initial study. Biologists could rig these various control elements so that the virus emits different colored fluorescent markers when it encounters cells that have different protein attributes.
"I think we can get at least another level or two of sophistication in the circuitry mapping," said Enquist.
In addition to Enquist, Tomishima, DeFalco, and Friedman,
co-authors on the Science paper are Hongyan Liu, Connie Zhao
and XiaoLi Cai of Rockefeller and Jamey Marth of the
University of California-San Diego. Enquist's work is
supported in large part by a grant from the National
Institutes of Health.