Cortical circuitry is differentially influenced by both excitatory and inhibitory neurons. More and more, researchers are learning that cortical inhibition makes an important and unique contribution to complex neural interplay, but traditional tracing methods are unable to distinguish between these two types of cells. Now, researchers from the University of California Irvine (UCI) have developed a new technique for retrograde labeling of either excitatory or inhibitory neural inputs using a combination of neurotropic vectors, specific cell type promoters, and a specially engineered rabies virus. Their study was published in the September 23, 2013 issue of Current Biology.
“The standard ways of tracing circuits are great if you have a brain region where there’s only one type of neuron. Then you’re all set,” said David Lyon, assistant professor of Anatomy and Neurobiology at UCI and lead author of the study. “But the neocortex has different types of cells all mixed up together, and we couldn’t target a particular one. That limited the kinds of questions we could ask about these circuits.”
By pairing a genetically modified rabies virus with neurotropic adeno-associated and lentiviral vectors and a promoter that only works in inhibitory cells, Lyon and colleagues were able to essentially turn an individual inhibitory neuron into a tracer.
“When delivering this rabies virus to a particular neuron, the rabies will replicate there and then spread to all the neurons around,” he said. “And whatever you deliver with the rabies is going to be expressed at extremely high levels very quickly.”
Lyon and colleagues used the technique to trace and label presynaptic connections in the visual cortex (V1) of mice and cats. They found a higher percentage of inputs to excitatory neurons than inhibitory neurons in this particular region. “We are using the technique to answer questions about early visual processing and how inhibitory cells shape orientation selectivity, which are precursors to shape, object and motion perception. The organization of these circuits is very complex, very complicated, and using this kind of technique, we can get a better idea of the basic organizational principles of V1.”
Lyon said the technique can also be used in other parts of the brain as well as in primate cortex, but he cautioned that the rabies virus’s speed of replication, which is what makes it such a phenomenal vector, also has a downside. Within about two weeks, the tracers start to kill the cells they infect. But despite its limitations, Lyon argued that the technique will help researchers understand the types of connections that make up a neural circuit.
“This [technique] is as accessible as traditional tracers (provided you are approved to use rabies virus), but it reaches a whole new level of detail,” he said. “And in addition to tracing, you can deliver genes for optogenetics and molecular imaging, which will allow you to look at the functional responses of cells in a circuit.”
Moving forward, Lyon and colleagues hope to piggyback this tracing method with other functional techniques to move beyond simply labeling neurons to studying how inhibitory and excitatory connections influence function.