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Deconstructing Brain Wiring, One Neuron at a Time

Imagine you were given a bowl of spaghetti then asked to draw a diagram of the contacts a single strand made with neighboring noodles. Sound challenging? Neuroscientists are faced with an even more gargantuan task in their effort to construct a wiring diagram of the connections made by billions of tangled neurons in the brain.

Edward Callaway, professor in the Systems Neurobiology Laboratories, and postdoctoral researcher Ian Wickersham reported in Neuron a method to make construction of that brain map feasible.

For decades researchers have attempted to devise a method, suggested by the late Francis Crick, in which a single neuron is injected with a substance that would stain all the neurons connected to it. The Salk group succeeded by infecting a single neuron – analogous to one noodle in the bowl – with a rabies virus carrying a gene for green fluorescing protein. The virus then spread to neurons directly connected to the first cell via their synapses – the gaps between neurons and their immediate neighbors – turning those cells green.

Developing this method required two different modifications of the rabies virus. The first trick was to devise a way to infect only a single cell. This was accomplished with a strategy developed by John Young, professor in the Infectious Disease Laboratory, and co-author on the study. The virus was coated with a protein from an avian virus so that it would only be able to infect cells expressing the avian viral receptor.

The second trick was to make a virus that could spread to directly connected cells, then stop so it could not continue to downstream cells. To accomplish this, researchers engineered the virus so that it was missing a gene required to cross a synapse. They then supplied both the avian virus receptor and the missing viral gene to neurons in rat brain tissues.

When they infected those tissues with the altered virus, the result was spectacular: selectively infected cells indeed transferred the virus to only their immediate neighbors, which could be identified by their brilliant green fluorescence. It was as if you could choose your target noodle and then magically light up only the noodles touching it.

This innovation will not only enable investigators to undertake functional studies of the way neurons affect their neighbors but it will also be invaluable in the effort to understand brain cell connectivity.