BASIC SCIENCE: Tracing Appetite Through the Brain

Applied Genetics News, April, 2001

Howard Hughes Medical Institute investigator Jeffrey Friedman and colleagues from several institutions have used pseudorabies virus to create a biological tracer that only propagates itself in neurons that express the leptin receptor or neuropeptide Y (NPY), an appetite-stimulating substance found in neurons. The virus, which travels upstream from the site of infection, jumping from neuron to neuron, was engineered to carry a gene for green fluorescent protein (GFP). The presence of the fluorescent protein enabled the scientists to trace the path of the virus as it moved through the brain. Their research is published in the March 30 issue of Science.

Leptin, discovered by Friedman in 1994, is produced by fat tissue and secreted into the bloodstream. The effect of the hormone is to cause fat loss and decreased appetite. In the brain, leptin affects food intake by acting on distinct classes of neurons in the hypothalamus that express the leptin receptor. However, that is not the whole story.

"It's obvious that the decision of whether or not to eat has some conscious input," notes Friedman. "For example, there is higher cortical input involved in making the decision about whether or not we're going to skip a meal, try to diet or eat less. While our study is only a beginning and doesn't address such behavioral issues, it's pretty clear that people differ in how much willpower they have," he says. "And willpower is not a metaphysical thing; it's a bunch of neural connections and neural circuits."

The scientists developed a viral tracing system that would label initially only those hypothalamic neurons expressing the leptin receptor, or those producing neuropeptide Y, an appetite- stimulating peptide found in abundance in certain types of neurons. The scientists found that they could achieve such specificity by building an "off" switch into the virus that was controlled by an enzyme called Cre recombinase. In the engineered virus, Cre is required for PRV to begin replicating.

The researchers generated two strains of mice in which the Cre recombinase enzyme was coexpressed in nerve cells that express either NPY or the leptin receptor. The virus was capable of replicating only in those cells expressing Cre. Since the virus expressed GFP, the presence of the virus was easy to trace.

The researchers next injected the Cre-dependent, GFP-tagged virus directly into a region of the hypothalamus called the arcuate nucleus, a brain region where NPY and the leptin receptor are expressed. After injection, the virus first spread throughout the cells of the arcuate nucleus itself, causing the neurons containing the Cre recombinase gene to glow green (from the GFP). The virus then spread from these cells to other cells that were in contact with it, and so on. As the virus infected each additional neuron it amplified itself, allowing the researchers to trace the pathway of neurons leaving the hypothalamus.

When the scientists examined slices of mouse brain treated with the virus, they could see which regions of the brain send neurons into the brain's areas known to regulate feeding behavior. "We could see inputs from a number of other regions to the hypothalamus, which is where basic drives for feeding are controlled," says Friedman. "We could see inputs from brain centers that control emotion and from others that receive olfactory inputs. We also saw inputs from centers in the mouse that are the equivalent of centers that control higher cortical or cognitive functions in humans."

Friedman and their colleagues are beginning studies using other PRV strains that can follow the connections downward from the higher levels, as well as combinations of viruses engineered with different markers to trace multiple pathways simultaneously. They also plan to explore the hierarchy of the circuitry by using advanced microscopy and computer systems to generate 3-D reconstructions of the labeled cells.