Neurons Glow in 'Brainbow'

The circuitry of the brain is being imaged as never before thanks to a multicolor fluorescent protein labeling technique called "Brainbow."

Brainbow allows researchers to tag neurons with roughly 90 distinct colors, a huge leap over the mere handful of shades possible with current fluorescent labeling. By permitting visual resolution of individual brightly colored neurons, this increase should greatly help scientists in charting the circuitry of the brain and nervous system.

Multicolor labeling of neurons in a 'Brainbow' transgenic mouse. Neurons of the hippocampus (dentate gyrus). (Confocal microscopy by Jean Livet)

The technique is described in the cover story of the Nov. 1 issue of the journal Nature by a team led by Harvard University's Jean Livet, Joshua R. Sanes, and Jeff W. Lichtman. Sanes and Lichtman are professors in the Department of Molecular and Cellular Biology and the Center for Brain Science; Livet is a postdoctoral researcher.

"In the same way that a television monitor mixes red, green, and blue to depict a wide array of colors, the combination of three or more fluorescent proteins in neurons can generate many different hues," said Lichtman. "There are few tools neuroscientists can use to tease out the wiring diagram of the nervous system; Brainbow should help us much better map out the brain and nervous system's complex tangle of neurons."

The resulting images could also help scientists identify how brain wiring goes awry in many different diseases. Brainbow could also help track the complicated development of the mammalian nervous system, currently understood only in general terms. This, in turn, could shed light on the origins of the many brain disorders that arise early in development.

This image shows a portion of cortex (layer 5) from a 'Brainbow' transgenic mouse. Neurons are randomly labeled with combinations of fluorescent proteins expressed at distinct levels. (Confocal microscopy by Jean Livet)

Drawing upon a mix of genetic tricks and special proteins that cause cells to glow, Brainbow uses a well-known genetic recombination system known as Cre/lox in a new way, to shuffle genes encoding green, yellow, orange, and red fluorescent proteins.

The researchers painstakingly assembled the Brainbow transgene from snippets of DNA, and inserted it into neuronal DNA. As they predicted, the cut-and-paste recombination occurred totally at random, in the process assigning scores of different colors to neurons. This variation makes neurons leap out from one another visually under ordinary confocal microscopy.

"The technique drives the cell to switch on fluorescent protein genes in neurons more or less at random," said Livet. "You can think of Brainbow almost like a slot machine in its generation of random outcomes, and Cre/lox is the hand pulling the lever over and over again."

Using Brainbow to look at mouse neural circuits over periods as long as 50 days, the researchers were able to observe some neural reorganization over time and to ascertain that Brainbow labeling is stable and long-lived. Livet, Sanes, Lichtman, and colleagues are now using Brainbow to scour the nervous system for new insights into its organization and function.

In 'Brainbow' transgenic mice, nerve cells randomly express fluorescent proteins of different colors. Combinations of these proteins label neurons with multiple distinct colors, allowing one to parse out their processes. In this image from a portion of the cerebellum, the multicolor labeling reveals the intricate meshwork created by 'mossy fiber' axons forming synapses in the area (large spots). (Confocal microscopy by Tamily A. Weissman)

"We've already used Brainbow to take a first peek at the nervous system of mice, and we've observed some very interesting, and previously unrecognized, patterns of neuron arrangement," said Sanes. "As far as understanding what we're seeing, we've only just scratched the surface."

Additional co-authors are Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, and Robyn A. Bennis, all of the Department of Molecular and Cellular Biology and Center for Brain Science.

The work was funded by the James S. McDonnell Foundation and the National Institutes of Health.
For more information, visit: www.harvard.edu