Technicolor brains

David L. Shenkenberg

Because almost all neurons are naturally translucent, individual neurons cannot be distinguished from each other without some kind of labeling. Existing labeling methods can mark neurons with only a few colors, but more colors could facilitate tracing neuronal connections. Now, the brains of novel genetically engineered “Brainbow” mice fluoresce with as many as 90 colors.

The brains of genetically engineered Brainbow mice look like works of Impressionist art, yet they could answer serious scientific questions. Confocal microscopy by Tamily A. Weissman. Images reprinted from Nature with permission of the researchers.

The mice were generated by postdoctoral fellow Jean Livet, principal investigators Joshua R. Sanes and Jeff W. Lichtman, and their colleagues at Harvard University in Cambridge, Mass. These mice enable the tracing of neuronal connections in adult brains as well as connections that form or that disappear during normal development or that are improperly organized in disorders such as Alzheimer’s disease, according to Lichtman. Eventually they could enable the mapping of neuronal connections throughout the entire brain.

Scientists who generated the mice believe that they could be used to trace neural circuits, finding connections that occur during normal development or improper connections that are associated with neurodegenerative disorders such as Alzheimer’s disease. Confocal microscopy by Ryan W. Draft.

Analogous to the way a painter or a television display creates multiple hues from the three primary colors, the colors emitted by the brains of the Brainbow mice result from the spectral mixing of red, green and blue fluorescent proteins. The brains contain several fluorescent protein variants that emit shades of the primary colors, such as “cherry” or “tomato” fluorescent proteins for red and cerulean fluorescent protein for blue.

The same strategies used to generate the Brainbow brains may be extended to other organs and other model organisms, fueling biological research in numerous disciplines. Confocal microscopy by Jean Livet.

The random combination of these variants in individual neurons accounts for the diversity of the emitted colors and is a result of the genetic engineering strategies that the researchers employed.

Rather than tagging specific proteins, which can disturb protein function, the fluorescent proteins exist freely in neurons. The Brainbow mice age, reproduce and behave normally.

Once the researchers created them, they investigated the potential of the mice as a research tool in studies detailed in the Nov. 1 issue of Nature. To do so, they used an Olympus confocal microscope to image brains of live anesthetized mice as well as fixed brain tissue and peripheral nerves targeting muscles, and they performed image reconstructions and time-lapse imaging, traced neurons and observed neuron interactions with neighboring non-neuronal cells.

To excite the fluorescent proteins in the nervous tissue, the researchers used a 440-nm photodiode laser for blue fluorescent proteins, the 515-nm line of an argon-ion laser for yellow fluorescent proteins, and a 561-nm photodiode laser or a 568-nm krypton laser for red fluorescent proteins, the lasers of which were all produced by Melles Griot of Carlsbad, Calif. Lichtman said that, to produce the best images possible, they used software to perform maximum intensity projections, linear unmixing and adjustments of contrast, brightness and gamma levels.

Not just for looks

When the researchers saw the mice brains, they were surprised at the beauty and number of the fluorescent colors. “It was jaw-dropping,” Lichtman said. They quantified them using The Mathworks’ Matlab, determining that there were approximately 89 distinct colors in axons throughout the brain. In a more specific location of the brain, the hippocampus, they found about 166 distinct colors in cell bodies.

The colors also can be used to trace neurons in three-dimensional reconstructions of confocal images. To do so, the researchers used Reconstruct -- public-domain software developed and maintained by John C. Fiala at Boston University. They traced mossy fiber axons and granule cells -- the postsynaptic targets of mossy fiber axons -- showing that Brainbow mice could be used to quantify the number of neurons that innervate postsynaptic cells.

They observed neurons interacting with glial cells, which are neighboring non-neuronal cells. Over intervals lasting four to 50 days, they performed time-lapse imaging of Schwann cells, a particular type of glial cell. They also imaged the same region of a peripheral nerve over six days and found that the fluorescence remained constant in terms of color and intensity. They reported that this showed that the Brainbow mice can be used in long-term studies of nerve cells.

The researchers also noted that analysis using the mice currently is limited by the number of colors and by the ability to resolve the colors. They worked with various red fluorescent proteins; however, the reds generally did not have the desired photostability or brightness, but new fluorescent proteins are being developed by laboratories across the globe. “Future Brainbow mice may use other colors,” Lichtman said.

The method uses a genetic element, called a promoter, to drive the expression of fluorescent proteins in mouse neurons. Lichtman said that changing the promoter could result in multicolor fluorescence in organs other than the brain and in other model organisms besides mice -- something of interest to researchers working with other organs and organisms. He also said that, although the promoter they used drives expression robustly in many neurons, it does not do so everywhere in the brain, and that changing the promoter could result in Brainbow mice that fluoresce multiple colors in other neuronal types. This strategy also may be used to label stem cells or cells that are involved in the immune response.

An image of a Brainbow mouse brain won first prize in the 2007 Olympus BioScapes competition. The researchers have made four strains of the prizewinning mice available through The Jackson Laboratory.