Fly Over the ‘Brainbow’

Two neural mapping techniques illuminate the delicate architecture of flies’ brains.

Four years ago, Harvard scientists devised a way to make mouse neurons glow in a breathtaking array of colors, a technique dubbed “Brainbow.” This allowed scientists to trace neurons’ long arms, known as the dendrites and axons, through the brain with incredible ease, revealing a map of neuron connections. 

Using a clever trick of genetic engineering, in which genes for three or more different fluorescent proteins were combined like paints to generate different hues, researchers created a system to make each neuron glow one of 100 different colors. The result was that the dendrites and axons of individual neurons, previously almost impossible to pick apart from their neighbors, could be traced through the mouse brain according to their color. 

Now, fruit fly researchers have a similar bonanza on their hands. Last week, two Brainbow-based methods for making fly neurons glow customized colors—called dBrainbow and Flybow—were published in Nature Methods. This is the first time that scientists have converted the technique to work in fruit flies, and because these organisms have a very sophisticated set of existing genetic tools, researchers can exert even greater control over when and where the fluorescent proteins are expressed. 

Because axons and dendrites are so long and fine, it’s hard to tell which neurons they are from. Researchers have traditionally had to stain just one or two neurons in each sample, painstakingly compiling data from many brains to build a map. In contrast, many neurons are easily discernible in this cross-section of a fly’s brain made using dBrainbow. Using dBrainbow images, Julie H. Simpson and colleagues at the Howard Hughes Medical Institute’s Janelia Farm could tell which motor neurons controlled parts of a fly’s proboscis, which it uses to take in food.

Both techniques have reduced the number of color options from the original brainbow—dBrainbow has six and Flybow, developed by Iris Salecker and colleagues at the National Institute for Medical Research in London, has four. This makes it easier to identify neurons. 

In dBrainbow, the color indicates which neurons arose from the same progenitor cell during development: each progenitor “decides” what color it will be, and all of its daughter cells will share that color, which is handy for studying how connections between different lineages of neurons are formed. In this shot of a fly’s head, different progenitors gave rise to the blue olfactory neurons on the right and the red olfactory neurons on the left.

In contrast, Flybow cells can be made to “decide” their color at any point in development, because the enzymatic process that causes them to change colors is activated by applying heat. The cells are engineered so their default color is green. The longer they are heated, the more cells will switch from green to blue, yellow, or red. Heat applied early in development produces an effect similar to dBrainbow, while heat applied later produces individual cells that each glow their own color. Here, the visual system of an adult fruit fly shows individual neurons in four colors.

Using existing genetic techniques, scientists can restrict the activation of the dBrainbow and Flybow genes to specific subsets of cells, so only the neurons relevant to their research are visible. In this dBrainbow image, a group of about 2,000 highly studied neurons thought to underlie male courtship behavior are colored according to different subpopulations.

In a typical study, the red, yellow, and blue neurons in this image of a developing fly’s nerve cord would never be seen together, but would instead be spread across many samples, like the pieces of a jigsaw puzzle, leaving scientists to imagine what they might look like in the intact fly. 

“It is a real revelation to see them actually next to each other, at the same time,” says Salecker. “To see them as they are, with their neighbors—it makes a huge difference.”


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