Splashes of fuchsia, streaks of crimson and a smattering of taupe. When these dazzling displays of color—each hue denoting a different neuron—first appeared in the neuroscientific community in 2007, researchers hailed them as a novel way to understand brain structure. By inserting genes from bacteria, corals and jellyfish to code for three different fluorescent proteins into mouse nerve cells, Harvard University neuroscientists created neurons that would express a random combination of the proteins. These combinations could illuminate cells in up to 90 distinct colors, transforming scientific images into visually striking works of art.

Originally developed to map neural circuits, today some scientists believe that the technique, called brainbow, is far better suited to other tasks, such as offering a closer look at what specific neurons do and at the brain cells behind behaviors. And because the daughter cells of brainbow neurons inherit the same color as their parents, scientists can use this method to study how clusters of cells grow and develop. This collection of images gives us a glimpse into these recent discoveries.

PRIVATE LIVES OF FLIES
In the fruit fly brain shown above, scientists selectively labeled neurons that release octopamine, a neurochemical involved in several behaviors, including sleep and aggression.

The image at the right reveals some 2,000 neurons that play a role in a male fruit fly's courtship behavior.

CREDIT: COURTESY OF PHUONG CHUNG AND JULIE H. SIMPSON
WINDOW INTO DEVELOPMENT
Zebra fish embryos are small and see-through, which allows scientists to view living animals during early development under a microscope. The near left image provides a close-up view of a ventricle in a zebra fish brain, a cavity where new neurons are born. Each uniformly colored, vertically extending line includes neurons originating from the same parent cell. The far left image provides a view of a fish's body from above the spine.

Credit: ZACHARY TOBIAS, ZEBRA FISH VENTRICLE PHOTOGRAPHED AT TAMILY A. WEISSMAN'S LABORATORY AT LEWIS & CLARK COLLEGE OF ARTS & SCIENCES (zebra fish neurons) Courtesy of ALBERT PANMedical College of Georgia, Georgia Regents University (zebra fish body)
FIBER-OPTIC
In this mouse optic nerve, the multicolored cells are oligodendrocytes, which form the myelin sheath, a fatty protective layer that insulates nerves. In this nerve, it shields electrical signals between the eyes and brain.

Credit: COURTESY OF ALAIN CHÉDOTAL Vision Institute, INSERM

ORIGINAL BRAINBOW NEURONS
The image above, showing human embryonic kidney cells, was one of the first proof-of-concept demonstrations of brainbow, published in 2007.

CREDIT: FROM “TRANSGENIC STRATEGIES FOR COMBINATORIAL EXPRESSION OF FLUORESCENT PROTEINS IN THE NERVOUS SYSTEM,” BY JEAN LIVET ET AL., IN NATURE, VOL. 450; NOVEMBER 1, 2007
STARRY CORTEX
Multicolored pyramidal neurons illuminate a mouse cortex at the left. These neurons are among the most common type of cell in the mammalian brain and are believed to be involved in complex kinds of cognition.

CREDIT: FROM “MULTIPLEX CELL AND LINEAGE TRACKING WITH COMBINATORIAL LABELS,” BY KARINE LOULIER ET AL., IN NEURON, VOL. 81, NO. 3; FEBRUARY 5, 2014 (SUPPLEMENTAL INFORMATION)
GIANT JUNCTIONS
The limits of current technology make it hard to see the synaptic connections between neurons using brainbow. One exception is the neuromuscular junction—shown in mice in the two images at the right above—where the neurons that connect with muscle fibers are large and few in number. Each ribbon of color is a motor neuron that competes with its neighbors until only one remains at the synapse. Scientists can combine these images to construct a more complete view of a motor neuron circuit, as depicted in the left image.

CREDIT: COURTESY OF IAN BOOTHBY AND JEFF W. LICHTMAN Harvard University
CHICKEN OR EGG?
Scientists also use brainbow-based techniques to study neural development in chicks. In the far left image, researchers labeled neurons in an area near the front of the developing brain of an 11-day-old chick embryo. The near left image depicts neurogenesis—or the growth of new neurons—in a chick's spinal cord. Daughter cells match the color of the parent cell.

CREDIT: FROM “CLONE IS A NEW METHOD TO TARGET SINGLE PROGENITORS AND STUDY THEIR PROGENY IN MOUSE AND CHICK,” BY FERNANDO GARCÍA-MORENO ET AL., INDEVELOPMENT, VOL. 141, NO. 7; APRIL 1, 2014 (chick brain neurons); FROM “MULTIPLEX CELL AND LINEAGE TRACKING WITH COMBINATORIAL LABELS,” BY KARINE LOULIER ET AL., INNEURON, VOL. 81, NO. 3; FEBRUARY 5, 2014 (chick spinal cord neurons)